Brush-arm star polymer imaging agents and uses thereof

ABSTRACT

Disclosed are methods, compositions, reagents, systems, and kits to prepare nitroxide-functionalized brush-arm star polymer organic radical contrast agent (BASP-ORCA) as well as compositions and uses thereof. Various embodiments show that BASP-ORCA display unprecedented per-nitroxide and per-molecule transverse relaxivities for organic radical contrast agents, exceptional stability, high water solubility, low in vitro and in vivo toxicity, and long blood compartment half-life. These materials have the potential to be adopted for tumor imaging using clinical high-field 1H MRI techniques.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application, U.S. Ser. No. 62/528,026, filed Jun. 30, 2017, which is incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under grant numbers R01 EB019950 and R21 EB018529 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Among the many imaging modalities for medical diagnostics, magnetic resonance imaging (MRI) is one of the most useful due to its ability to non-invasively generate three-dimensional detailed anatomical images with high spatial resolution while not requiring an ionizing source and remaining insensitive to depth.^(1,2,3,4) Current clinical MRI methods depict the spatial distribution and chemical environment of water protons (¹H) within a region of interest (ROI) with the use of contrast agents. These contrast agents are divided into two primary classes: T₁ contrast agents (e.g., paramagnetic metals such as gadolinium or manganese) that afford positive-contrast images primarily by locally reducing the protons' longitudinal relaxation time (spin-lattice, T₁), and T₂ contrast agents (e.g., superparamagnetic iron oxide nanoparticles) that afford negative-contrast images by locally reducing the transverse relaxation time (spin-spin, T₂) of water molecules.^(5,6) The corresponding ¹H water relaxivities (r₁ and r₂, respectively) of a contrast agent characterize the extent to which the agent decreases the T₁ and T₂ times of water. Contrast agents with greater r₁ and r₂ values provide increased image contrast compared to those with lower values at the same concentration.^(6,7)

Most MRI contrast agents with large r₁ and/or r₂ values contain metals that feature a large number of unpaired electrons. For example, small molecule^(8,9,10,11,12,13) and nanoparticle-based^(14,15,16,17,18,19,20,21) contrast agents featuring Gd, Mn, Fe-oxide, and other metals have been reported to function as either T₁ or T₂ contrast agents or both. Furthermore, metal-based contrast agents that display advanced functions such as multimodal imaging,^(8,9,10,12,13,17,20,21) enhanced target-specific accumulation,^(14,18,19) or sensing^(8,11,12,13,14) have all been developed. Metal-based contrast agents, especially nanoparticle systems which tend to accumulate in biological tissues, suffer from a few key limitations. Most notably, they could present toxicity concerns in patients with hindered kidney function and newborn children; additionally, Gd-based agents, perhaps the most widely used contrast agents in the clinic, have recently been linked to a rising prevalence of toxic Gd ions in the environment.^(5,22,23,24,25,26,27,28,29,30) Thus, there is interest in developing “metal-free” MRI contrast agents that make use of organic components. Such agents could enable MRI in at-risk patient populations, and they could potentially open new avenues for functional/responsive MRI based on in vivo organic transformations that require longer timeframes and contrast agents with low toxicity. For example, organic nanoparticles could have advantages for imaging strategies that require long-term tissue accumulation, such as tumor imaging.

SUMMARY OF THE INVENTION

Four main classes of metal-free MRI contrast agents have been most widely studied: paramagnetic nitroxide-based Organic Radical Contrast Agents (ORCAs), hyperpolarized ¹³C agents, ¹⁹F MRI contrast agents, and chemical exchange saturation transfer (CEST) contrast agents. While ¹⁹F MRI and CEST agents have undergone many advances in recent years,^(31,32,33,34,35,36,37,38) these approaches often suffer from low sensitivity, and in some cases, require a high contrast agent concentration (10-50 mM), long imaging times, and/or potentially harmful high-intensity radio-frequency fields. Hyperpolarized ¹³C agents, on the other hand, can theoretically afford up to 10⁵ sensitivity improvements; nevertheless, issues including short hyperpolarization lifetimes that lead to limited imaging times and complexity in terms of the chemistry and instrumentation required for generation of the hyperpolarized agent remain major challenges.^(39,40,41) Furthermore, neither ¹⁹F MRI, CEST, nor hyperpolarized ¹³C agents have become common in the clinic, which could prevent their rapid adoption.^(39,40,41,42,43,44,45,46) In contrast, nitroxide ORCAs rely on standard water relaxation mechanisms to achieve MRI contrast, and therefore, could be immediately translated to clinical applications. However, several key challenges limit the clinical feasibility of nitroxide ORCAs. First, the nitroxide radical only possesses one unpaired electron. As a result, compared to metal-based contrast agents such as Gd³⁺ (7 unpaired electrons) or Mn²⁺ (5 unpaired electrons), nitroxide ORCAs inherently suffer from much lower ¹H water relaxivity. One strategy to overcome this limitation is to use a poly(nitroxide) macromolecule where the relatively low per nitroxide relaxivity is multiplied by the number of nitroxide moieties bound to the macromolecule to achieve higher relaxivity. The second major limitation of nitroxide ORCAs is the fact that they are typically reduced rapidly in vivo to diamagnetic hydroxylamines, thus rendering them ineffective as contrast agents shortly after injection.^(47,48,49,50) Indeed, initial efforts to utilize nitroxides as MRI contrast agents exposed these shortcomings,^(51,52) and though their rapid bioreduction has been cleverly exploited to enable redox-mapping in vitro and in vivo,^(53,54,55,56,57) an in vivo-stable nitroxide ORCA that allows for longitudinal studies over clinically meaningful timescales following systemic administration has yet to be developed.

Nanoparticle-based nitroxide ORCAs with long-term in vivo stability could be particularly useful for tumor imaging; nanoparticles of suitable size (˜10-200 nm) are known to passively accumulate in tumors via the enhanced permeation and retention effect, especially in common murine (e.g. mouse) models, but hours to tens of hours are often needed to reach maximal accumulation.^(58,59,60,61,62,63,64) As stated above, there are no nitroxide-based molecules or materials with demonstrated capability to provide in vivo MRI contrast after such long times. This problem is exacerbated in murine models where imaging is often used for preclinical studies of disease development; murine tissues contain higher levels of metabolic antioxidants, which lead to faster nitroxide reduction rates.^(65,66) Thus, the development of stable nitroxide-based ORCAs with high relaxivities would open a new class of imaging applications whereby the accumulation of contrast agents in tissues could enable MRI without toxicity concerns.^(50,67,68) Moreover, the flexibility of nanoparticle-based materials could facilitate future image-guided drug delivery strategies.

Compositions, methods, systems, and kits that allow for the preparation and use of a new class of Brush-Arm Star Polymer ORCAs (BASP-ORCAs) are disclosed herein. In addition, in vitro and in vivo experiments utilizing BASP-ORCAs are described along with compositions, methods, systems, and uses thereof. BASP-ORCAs are designed to overcome the aforementioned challenges associated with MRI using typical contrast agents.

In certain embodiments, the present disclosure provides a brush-star arm polymer with an associated imaging agent (e.g., a nitroxide-based contrast agent). Such a polymer may be in the form of a particle, such as a nanoparticle. In certain embodiments, the present disclosure provides a brush-arm star polymer with an imaging agent comprising at least 100 repeating units selected from Formula (I) and Formula (II):

and salts thereof, wherein:

each of A, A¹, and B is independently C₁-C₁₂ alkylene, C₂-C₁₂ alkenylene, C₂-C₁₂ alkynylene, or C₁-C₁₂ heteroalkylene, C₂-C₁₂ heteroalkenylene, C₂-C₁₂ heteroalkynylene, wherein each alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene is optionally substituted with 1-24 independently selected R¹;

X is an imaging agent as described herein;

P is alkylene, heteroalkylene, or polymer;

L is a bond, —O—, —S—, —S—S—, C₁-C₁₂ alkylene, C₂-C₁₂ alkenylene, C₂-C₁₂ alkynylene, C₁-C₁₂ heteroalkylene, (C₀-C₁₂ alkylene)-arylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-arylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ alkylene)-arylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ heteroalkylene)-arylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ alkylene)-heteroarylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heteroarylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heteroarylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ alkylene)-heterocyclylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heterocyclylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-aryl-(C₀-C₁₂ heteroalkylene), or (C₀-C₁₂ heteroalkylene)-heterocyclylene-(C₀-C₁₂ heteroalkylene), wherein each alkylene, alkenylene, alkynylene, heteroalkylene, arylene, heteroarylene, or heterocyclylene is optionally substituted with 1-24 independently selected R¹, and combinations thereof;

each R¹ is independently alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR^(A), —N(R^(A))₂, —NR^(A)C(O)R^(A), —NR^(A)C(O)OR^(A), —NR^(A)C(O)N(R^(A))₂, —C(O)N(R^(A))₂, —C(O)R^(A), —C(O)OR^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂, —SR^(A), or —S(O)_(m)R^(A);

each R^(A) is independently hydrogen, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, or C₁-C₆ haloalkyl;

each of a and b are independently an integer between 1 and 10000, inclusive;

each of “1”, “2”, “3”, “4”, “5”, and “6” is independently a terminal group selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted acyl, optionally substituted hydroxyl, optionally substituted amino, and optionally substituted thiol; or represents a bond to a another repeating unit of Formula (I) or Formula (II);

y is an integer between 1 and 100, inclusive; and

m is 1 or 2.

In certain embodiments, the imaging agent is a chelated metal, inorganic compound, organometallic compound, organic compound, or salt thereof. In certain embodiments, the imaging agent is an organic compound. In certain embodiments, the imaging agent is an organic radical. In certain embodiments, the imaging agent is a nitroxide-containing imaging agent. In certain embodiments, the imaging agent is

In certain embodiments, the imaging agent is useful for performing magnetic resonance imaging.

In certain embodiments, the imaging agent is a salt of an organic compound. In certain embodiments, the imaging agent is

In certain embodiments, the imaging agent is useful for performing near-infrared fluorescence imaging.

In certain embodiments, the present disclosure provides methods to produce a brush-arm star polymer comprising reacting one or more macromonomers associated with an imaging agent with a metathesis catalyst to form a living polymer; and mixing a crosslinker with the living polymer. In certain embodiments, at least two different macromonomers each containing a different imaging agents are reacted together to form the brush-arm star polymer.

In certain embodiments, the macromonomer is of Formula (III):

or a salt thereof, wherein:

each of A, A¹, and B is independently C₁-C₁₂ alkylene, C₂-C₁₂ alkenylene, C₂-C₁₂ alkynylene, or C₁-C₁₂ heteroalkylene, C₂-C₁₂ heteroalkenylene, C₂-C₁₂ heteroalkynylene, wherein each alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene is optionally substituted with 1-24 independently selected R¹;

X is an imaging agent described herein;

P is alkylene, heteroalkylene, or polymer;

L is a bond, —O—, —S—, —S—S—, C₁-C₁₂ alkylene, C₂-C₁₂ alkenylene, C₂-C₁₂ alkynylene, C₁-C₁₂ heteroalkylene, (C₀-C₁₂ alkylene)-arylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-arylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ alkylene)-arylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ heteroalkylene)-arylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ alkylene)-heteroarylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heteroarylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heteroarylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ alkylene)-heterocyclylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heterocyclylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-aryl-(C₀-C₁₂ heteroalkylene), or (C₀-C₁₂ heteroalkylene)-heterocyclylene-(C₀-C₁₂ heteroalkylene), wherein each alkylene, alkenylene, alkynylene, heteroalkylene, arylene, heteroarylene, or heterocyclylene is optionally substituted with 1-24 independently selected R¹, and combinations thereof;

each R¹ is independently alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR^(A), —N(R^(A))₂, —NR^(A)C(O)R^(A), —NR^(A)C(O)OR^(A), —NR^(A)C(O)N(R^(A))₂, —C(O)N(R^(A))₂, —C(O)R^(A), —C(O)OR^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂, —SR^(A), or —S(O)_(m)R^(A);

each R^(A) is independently hydrogen, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, or C₁-C₆ haloalkyl;

y is an integer between 1 and 100, inclusive; and

m is 1 or 2.

In certain embodiments, the macromonomer is of the formula:

In certain embodiments, the macromonomer is of the formula:

In certain embodiments, the present disclosure describes compositions comprising a polymer described herein (i.e., BASP-ORCAs). In certain embodiments, the composition further comprises a pharmaceutically acceptable excipient.

In certain embodiments, the present disclosure describes kits comprising a polymer described herein (i.e., BASP-ORCAs), or a composition comprising a polymer (i.e., BASP-ORCAs), and instructions for use.

In certain embodiments, the present disclosure provides methods of imaging a subject, the method comprising the steps of: administering to a subject a polymer described herein (i.e., BASP-ORCAs), or a composition comprising a polymer described herein (i.e., BASP-ORCAs); and acquiring an image of at least a portion of the subject. In certain embodiments, the imaging modality is magnetic resonance imaging. In certain embodiments, the imaging modality is near-infrared fluorescence imaging.

In certain embodiments, the present disclosure provides compounds, polymers, particles, nanoparticles, compositions, and kits described herein for use in a method of the present disclosure.

The details of certain embodiments of the invention are set forth in the Detailed Description of Certain Embodiments, as described below. Other features, objects, and advantages of the invention will be apparent from the Definitions, Examples, Figures, and Claims.

Definitions

Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5^(th) Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3^(rd) Edition, Cambridge University Press, Cambridge, 1987.

Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various stereoisomeric forms, e.g., enantiomers and/or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, N Y, 1962); and Wilen, S. H., Tables of Resolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, Ind. 1972). The invention additionally encompasses compounds as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

In a formula,

is a single bond where the stereochemistry of the moieties immediately attached thereto is not specified, - - - is absent or a single bond, and - - - or - - - is a single or double bond.

Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, replacement of ¹⁹F with ¹⁸F, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbon are within the scope of the disclosure. Such compounds are useful, for example, as analytical tools or probes in biological assays.

When a range of values is listed, it is intended to encompass each value and subrange within the range. For example “C₁₋₆ alkyl” is intended to encompass, C₁, C₂, C₃, C₄, C₅, C₆, C₁₋₆, C₁₋₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆, C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄, C₄₋₆, C₄₋₅, and C₅₋₆ alkyl.

The term “aliphatic” refers to alkyl, alkenyl, alkynyl, and carbocyclic groups. Likewise, the term “heteroaliphatic” refers to heteroalkyl, heteroalkenyl, heteroalkynyl, and heterocyclic groups.

The term “alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 10 carbon atoms (“C₁₋₁₀ alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C₁₋₉ alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C₁₋₈ alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C₁₋₇ alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C₁₋₆ alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C₁₋₅ alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C₁₋₄ alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C₁₋₃ alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C₁₋₂ alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“C₁ alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C₂₋₆ alkyl”). Examples of C₁₋₆ alkyl groups include methyl (C₁), ethyl (C₂), propyl (C₃) (e.g., n-propyl, isopropyl), butyl (C₄) (e.g., n-butyl, tert-butyl, sec-butyl, iso-butyl), pentyl (C₅) (e.g., n-pentyl, 3-pentanyl, amyl, neopentyl, 3-methyl-2-butanyl, tertiary amyl), and hexyl (C₆) (e.g., n-hexyl). Additional examples of alkyl groups include n-heptyl (C₇), n-octyl (C₈), and the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one or more substituents (e.g., halogen, such as F). In certain embodiments, the alkyl group is an unsubstituted C₁₋₁₀ alkyl (such as unsubstituted C₁₋₆ alkyl, e.g., —CH₃ (Me), unsubstituted ethyl (Et), unsubstituted propyl (Pr, e.g., unsubstituted n-propyl (n-Pr), unsubstituted isopropyl (i-Pr)), unsubstituted butyl (Bu, e.g., unsubstituted n-butyl (n-Bu), unsubstituted tert-butyl (tert-Bu or t-Bu), unsubstituted sec-butyl (sec-Bu or s-Bu), unsubstituted isobutyl (i-Bu)). In certain embodiments, the alkyl group is a substituted C₁₋₁₀ alkyl (such as substituted C₁₋₆ alkyl, e.g., —CH₂F, —CHF₂, —CF₃ or benzyl (Bn)).

The term “haloalkyl” is a substituted alkyl group, wherein one or more of the hydrogen atoms are independently replaced by a halogen, e.g., fluoro, bromo, chloro, or iodo. “Perhaloalkyl” is a subset of haloalkyl, and refers to an alkyl group wherein all of the hydrogen atoms are independently replaced by a halogen, e.g., fluoro, bromo, chloro, or iodo. In some embodiments, the haloalkyl moiety has 1 to 8 carbon atoms (“C₁₋₈ haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 6 carbon atoms (“C₁₋₆ haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 4 carbon atoms (“C₁₋₄ haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 3 carbon atoms (“C₁₋₃ haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 2 carbon atoms (“C₁₋₂ haloalkyl”). In some embodiments, all of the haloalkyl hydrogen atoms are replaced with fluoro to provide a perfluoroalkyl group. In some embodiments, all of the haloalkyl hydrogen atoms are replaced with chloro to provide a “perchloroalkyl” group. Examples of haloalkyl groups include —CF₃, —CF₂CF₃, —CF₂CF₂CF₃, —CCl₃, —CFCl₂, —CF₂Cl, and the like.

The term “heteroalkyl” refers to an alkyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkyl group refers to a saturated group having from 1 to 10 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC₁₋₁₀ alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 9 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC₁₋₉ alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 8 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC₁₋₈ alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 7 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC₁₋₇ alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 6 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC₁₋₆ alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 5 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroC₁₋₅ alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 4 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroC₁₋₄ alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 3 carbon atoms and 1 heteroatom within the parent chain (“heteroC₁₋₃ alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 2 carbon atoms and 1 heteroatom within the parent chain (“heteroC₁₋₂ alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 carbon atom and 1 heteroatom (“heteroC₁ alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 2 to 6 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroC₂₋₆ alkyl”). Unless otherwise specified, each instance of a heteroalkyl group is independently unsubstituted (an “unsubstituted heteroalkyl”) or substituted (a “substituted heteroalkyl”) with one or more substituents. In certain embodiments, the heteroalkyl group is an unsubstituted heteroC₁₋₁₀ alkyl. In certain embodiments, the heteroalkyl group is a substituted heteroC₁₋₁₀ alkyl.

The term “alkenyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 10 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). In some embodiments, an alkenyl group has 2 to 9 carbon atoms (“C₂₋₉ alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C₂₋₈ alkenyl”). In some embodiments, an alkenyl group has 2 to 7 carbon atoms (“C₂₋₇ alkenyl”). In some embodiments, an alkenyl group has 2 to 6 carbon atoms (“C₂₋₆ alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C₂₋₅ alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C₂₋₄ alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C₂₋₃ alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C₂ alkenyl”). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C₂₋₄ alkenyl groups include ethenyl (C₂), 1-propenyl (C₃), 2-propenyl (C₃), 1-butenyl (C₄), 2-butenyl (C₄), butadienyl (C₄), and the like. Examples of C₂₋₆ alkenyl groups include the aforementioned C₂₋₄ alkenyl groups as well as pentenyl (C₅), pentadienyl (C₅), hexenyl (C₆), and the like. Additional examples of alkenyl include heptenyl (C₇), octenyl (C₈), octatrienyl (C₈), and the like. Unless otherwise specified, each instance of an alkenyl group is independently unsubstituted (an “unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with one or more substituents. In certain embodiments, the alkenyl group is an unsubstituted C₂₋₁₀ alkenyl. In certain embodiments, the alkenyl group is a substituted C₂₋₁₀ alkenyl. In an alkenyl group, a C═C double bond for which the stereochemistry is not specified (e.g., —CH═CHCH₃,

may be in the (E)- or (Z)-configuration.

The term “heteroalkenyl” refers to an alkenyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkenyl group refers to a group having from 2 to 10 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC₂₋₁₀ alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 9 carbon atoms at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC₂₋₉ alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 8 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC₂₋₈ alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 7 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC₂₋₇ alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 6 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC₂₋₆ alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 5 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“heteroC₂₋₅ alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 4 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“heteroC₂₋₄ alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 3 carbon atoms, at least one double bond, and 1 heteroatom within the parent chain (“heteroC₂₋₃ alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 6 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“heteroC₂₋₆ alkenyl”). Unless otherwise specified, each instance of a heteroalkenyl group is independently unsubstituted (an “unsubstituted heteroalkenyl”) or substituted (a “substituted heteroalkenyl”) with one or more substituents. In certain embodiments, the heteroalkenyl group is an unsubstituted heteroC₂₋₁₀ alkenyl. In certain embodiments, the heteroalkenyl group is a substituted heteroC₂₋₁₀ alkenyl.

The term “alkynyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 10 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) (“C₂₋₁₀ alkynyl”). In some embodiments, an alkynyl group has 2 to 9 carbon atoms (“C₂₋₉ alkynyl”). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (“C₂₋₈ alkynyl”). In some embodiments, an alkynyl group has 2 to 7 carbon atoms (“C₂₋₇ alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C₂₋₆ alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C₂₋₅ alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C₂₋₄ alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C₂₋₃ alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C₂ alkynyl”). The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples of C₂₋₄ alkynyl groups include, without limitation, ethynyl (C₂), 1-propynyl (C₃), 2-propynyl (C₃), 1-butynyl (C₄), 2-butynyl (C₄), and the like. Examples of C₂₋₆ alkenyl groups include the aforementioned C₂₋₄ alkynyl groups as well as pentynyl (C₅), hexynyl (C₆), and the like. Additional examples of alkynyl include heptynyl (C₇), octynyl (C₈), and the like. Unless otherwise specified, each instance of an alkynyl group is independently unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents. In certain embodiments, the alkynyl group is an unsubstituted C₂₋₁₀ alkynyl. In certain embodiments, the alkynyl group is a substituted C₂₋₁₀ alkynyl.

The term “heteroalkynyl” refers to an alkynyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkynyl group refers to a group having from 2 to 10 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC₂₋₁₀ alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 9 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC₂₋₉ alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 8 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC₂₋₈ alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 7 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC₂₋₇ alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC₂₋₆ alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 5 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“heteroC₂₋₅ alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 4 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“heteroC₂₋₄ alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 3 carbon atoms, at least one triple bond, and 1 heteroatom within the parent chain (“heteroC₂₋₃ alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“heteroC₂₋₆ alkynyl”). Unless otherwise specified, each instance of a heteroalkynyl group is independently unsubstituted (an “unsubstituted heteroalkynyl”) or substituted (a “substituted heteroalkynyl”) with one or more substituents. In certain embodiments, the heteroalkynyl group is an unsubstituted heteroC₂₋₁₀ alkynyl. In certain embodiments, the heteroalkynyl group is a substituted heteroC₂₋₁₀ alkynyl.

The term “carbocyclyl” or “carbocyclic” refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 14 ring carbon atoms (“C₃₋₁₄ carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a carbocyclyl group has 3 to 10 ring carbon atoms (“C₃₋₁₀ carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms (“C₃₋₈ carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms (“C₃₋₇ carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms (“C₃₋₆ carbocyclyl”). In some embodiments, a carbocyclyl group has 4 to 6 ring carbon atoms (“C₄₋₆ carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 6 ring carbon atoms (“C₅₋₆ carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms (“C₅₋₁₀ carbocyclyl”). Exemplary C₃₋₆ carbocyclyl groups include, without limitation, cyclopropyl (C₃), cyclopropenyl (C₃), cyclobutyl (C₄), cyclobutenyl (C₄), cyclopentyl (C₅), cyclopentenyl (C₅), cyclohexyl (C₆), cyclohexenyl (C₆), cyclohexadienyl (C₆), and the like. Exemplary C₃₋₈ carbocyclyl groups include, without limitation, the aforementioned C₃₋₆ carbocyclyl groups as well as cycloheptyl (C₇), cycloheptenyl (C₇), cycloheptadienyl (C₇), cycloheptatrienyl (C₇), cyclooctyl (C₈), cyclooctenyl (C₈), bicyclo[2.2.1]heptanyl (C₇), bicyclo[2.2.2]octanyl (C₈), and the like. Exemplary C₃₋₁₀ carbocyclyl groups include, without limitation, the aforementioned C₃₋₈ carbocyclyl groups as well as cyclononyl (C₉), cyclononenyl (C₉), cyclodecyl (C₁₀), cyclodecenyl (C₁₀), octahydro-1H-indenyl (C₉), decahydronaphthalenyl (C₁₀), spiro[4.5]decanyl (C₁₀), and the like. As the foregoing examples illustrate, in certain embodiments, the carbocyclyl group is either monocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g., containing a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) or tricyclic system (“tricyclic carbocyclyl”)) and can be saturated or can contain one or more carbon-carbon double or triple bonds. “Carbocyclyl” also includes ring systems wherein the carbocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on the carbocyclyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring system. Unless otherwise specified, each instance of a carbocyclyl group is independently unsubstituted (an “unsubstituted carbocyclyl”) or substituted (a “substituted carbocyclyl”) with one or more substituents. In certain embodiments, the carbocyclyl group is an unsubstituted C₃₋₁₄ carbocyclyl. In certain embodiments, the carbocyclyl group is a substituted C₃₋₁₄ carbocyclyl.

In some embodiments, “carbocyclyl” is a monocyclic, saturated carbocyclyl group having from 3 to 14 ring carbon atoms (“C₃₋₁₄ cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 10 ring carbon atoms (“C₃₋₁₀ cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“C₃₋₈ cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C₃₋₆ cycloalkyl”). In some embodiments, a cycloalkyl group has 4 to 6 ring carbon atoms (“C₄₋₆ cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C₅₋₆ cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C₅₋₁₀ cycloalkyl”). Examples of C₅₋₆ cycloalkyl groups include cyclopentyl (C₅) and cyclohexyl (C₅). Examples of C₃₋₆ cycloalkyl groups include the aforementioned C₅₋₆ cycloalkyl groups as well as cyclopropyl (C₃) and cyclobutyl (C₄). Examples of C₃₋₈ cycloalkyl groups include the aforementioned C₃₋₆ cycloalkyl groups as well as cycloheptyl (C₇) and cyclooctyl (C₈). Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted (an “unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more substituents. In certain embodiments, the cycloalkyl group is an unsubstituted C₃₋₁₄ cycloalkyl. In certain embodiments, the cycloalkyl group is a substituted C₃₋₁₄ cycloalkyl. In certain embodiments, the carbocyclyl includes 0, 1, or 2 C═C double bonds in the carbocyclic ring system, as valency permits.

The term “heterocyclyl” or “heterocyclic” refers to a radical of a 3- to 14-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“3-14 membered heterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or polycyclic (e.g., a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”) or tricyclic system (“tricyclic heterocyclyl”)), and can be saturated or can contain one or more carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. Unless otherwise specified, each instance of heterocyclyl is independently unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a “substituted heterocyclyl”) with one or more substituents. In certain embodiments, the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3-14 membered heterocyclyl. In certain embodiments, the heterocyclyl is substituted or unsubstituted, 3- to 7-membered, monocyclic heterocyclyl, wherein 1, 2, or 3 atoms in the heterocyclic ring system are independently oxygen, nitrogen, or sulfur, as valency permits.

In some embodiments, a heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In some embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

Exemplary 3-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azirdinyl, oxiranyl, and thiiranyl. Exemplary 4-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing 1 heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, dioxolanyl, oxathiolanyl and dithiolanyl. Exemplary 5-membered heterocyclyl groups containing 3 heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing 1 heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing 3 heteroatoms include, without limitation, triazinyl. Exemplary 7-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclyl groups containing 1 heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary bicyclic heterocyclyl groups include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, chromenyl, 1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl, 5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl, 5,7-dihydro-4H-thieno[2,3-c]pyranyl, 2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl, 4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl, 4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl, 4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl, 1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.

The term “aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 xt electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C₆₋₁₄ aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C₆ aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms (“C₁₀ aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms (“C₁₄ aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents. In certain embodiments, the aryl group is an unsubstituted C₆₋₁₄ aryl. In certain embodiments, the aryl group is a substituted C₆₋₁₄ aryl.

The term “heteroaryl” refers to a radical of a 5-14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-14 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings. “Heteroaryl” includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused polycyclic (aryl/heteroaryl) ring system. Polycyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl). In certain embodiments, the heteroaryl is substituted or unsubstituted, 5- or 6-membered, monocyclic heteroaryl, wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently oxygen, nitrogen, or sulfur. In certain embodiments, the heteroaryl is substituted or unsubstituted, 9- or 10-membered, bicyclic heteroaryl, wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently oxygen, nitrogen, or sulfur.

In some embodiments, a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In some embodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified, each instance of a heteroaryl group is independently unsubstituted (an “unsubstituted heteroaryl”) or substituted (a “substituted heteroaryl”) with one or more substituents. In certain embodiments, the heteroaryl group is an unsubstituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl group is a substituted 5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing 1 heteroatom include, without limitation, pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing 2 heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing 3 heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4 heteroatoms include, without limitation, tetrazolyl. Exemplary 6-membered heteroaryl groups containing 1 heteroatom include, without limitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing 2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4 heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing 1 heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include, without limitation, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl and phenazinyl.

“Heteroaralkyl” is a subset of “alkyl” and refers to an alkyl group substituted by a heteroaryl group, wherein the point of attachment is on the alkyl moiety.

The term “unsaturated bond” refers to a double or triple bond.

The term “unsaturated” or “partially unsaturated” refers to a moiety that includes at least one double or triple bond.

The term “saturated” refers to a moiety that does not contain a double or triple bond, i.e., the moiety only contains single bonds.

Affixing the suffix “-ene” to a group indicates the group is a divalent moiety, e.g., alkylene is the divalent moiety of alkyl, alkenylene is the divalent moiety of alkenyl, alkynylene is the divalent moiety of alkynyl, heteroalkylene is the divalent moiety of heteroalkyl, heteroalkenylene is the divalent moiety of heteroalkenyl, heteroalkynylene is the divalent moiety of heteroalkynyl, carbocyclylene is the divalent moiety of carbocyclyl, heterocyclylene is the divalent moiety of heterocyclyl, arylene is the divalent moiety of aryl, and heteroarylene is the divalent moiety of heteroaryl.

A group is optionally substituted unless expressly provided otherwise. The term “optionally substituted” refers to being substituted or unsubstituted. In certain embodiments, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups are optionally substituted. “Optionally substituted” refers to a group which may be substituted or unsubstituted (e.g., “substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or “unsubstituted” heteroalkyl, “substituted” or “unsubstituted” heteroalkenyl, “substituted” or “unsubstituted” heteroalkynyl, “substituted” or “unsubstituted” carbocyclyl, “substituted” or “unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or “unsubstituted” heteroaryl group). In general, the term “substituted” means that at least one hydrogen present on a group is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position. The term “substituted” is contemplated to include substitution with all permissible substituents of organic compounds, and includes any of the substituents described herein that results in the formation of a stable compound. The present invention contemplates any and all such combinations in order to arrive at a stable compound. For purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety. The invention is not intended to be limited in any manner by the exemplary substituents described herein.

Exemplary carbon atom substituents include, but are not limited to, halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(aa), —ON(R^(bb))₂, —N(R^(bb))₂, —N(R^(bb))₃ ⁺X⁻, —N(OR^(cc))R^(bb), —SH, —SR^(aa), —SSR^(cc), —C(═O)R^(aa), —CO₂H, —CHO, —C(OR^(cc))₂, —CO₂R^(aa), —OC(═O)R^(aa), —OCO₂R^(aa), —C(═O)N(R^(bb))₂, —OC(═O)N(R^(bb))₂, —NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa), —NR^(bb)C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —OC(═NR^(bb))R^(aa), —OC(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂, —OC(═NR^(bb))N(R^(bb))₂, —NR^(bb)(═NR^(bb))N(R^(bb))₂, —C(═O)NR^(bb)SO₂R^(aa), —NR^(bb)SO₂R^(aa), —SO₂N(R^(bb))₂, —SO₂R^(aa), —SO₂OR^(aa), —OSO₂R^(aa), —S(═O)R^(aa), —OS(═O)R^(aa), —Si(R^(aa))₃, —OSi(R^(aa))₃, —C(═S)N(R^(bb))₂, —C(═O)SR^(aa), —C(═S)SR^(aa), —SC(═S)SR^(aa), —SC(═O)SR^(aa), —OC(═O)SR^(aa), —SC(═O)OR^(aa), —SC(═O)R^(aa), —P(═O)₂R^(aa), —OP(═O)₂R^(aa), —P(═O)(R^(aa))₂, —OP(═O)(R^(aa))₂, —OP(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, —OP(═O)₂N(R^(bb))₂, —P(═O)(NR^(bb))₂, —OP(═O)(NR^(bb))₂, —NR^(bb)P(═O)(OR^(cc))₂, —NR^(bb)P(═O)(NR^(bb))₂, —P(R^(cc))₂, —P(R^(cc))₃, —OP(R^(cc))₂, —OP(R^(cc))₃, —B(R^(aa))₂, —B(OR^(cc))₂, —BR^(aa)(OR^(cc)), C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroC₁₋₁₀ alkyl, heteroC₂₋₁₀ alkenyl, heteroC₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

or two geminal hydrogens on a carbon atom are replaced with the group ═O, ═S, ═NN(R^(bb))₂, ═NNR^(bb)C(═O)R^(aa), ═NNR^(bb)C(═O)OR^(aa), ═NNR^(bb)S(═O)₂R^(aa), ═NR^(bb), or ═NOR^(cc);

each instance of R^(aa) is, independently, selected from C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroC₁₋₁₀ alkyl, heteroC₂₋₁₀alkenyl, heteroC₂₋₁₀alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(aa) groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(bb) is, independently, selected from hydrogen, —OH, —OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa), —C(═O)N(R^(cc))₂, —CO₂R^(aa), —SO₂R^(aa), —C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc), —SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc), —P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂, —P(═O)(NR^(cc))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroC₁₋₁₀alkyl, heteroC₂₋₁₀alkenyl, heteroC₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(bb) groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(cc) is, independently, selected from hydrogen, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroC₁₋₁₀ alkyl, heteroC₂₋₁₀ alkenyl, heteroC₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(cc) groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(dd) is, independently, selected from halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(ee), —ON(R^(ff))₂, —N(R^(ff))₂, —N(R^(ff))₃ ⁺X⁻, —N(OR^(ee))R^(ff), —SH, —SR^(ee), —SSR^(ee), —C(═O)R^(ee), —CO₂H, —CO₂R^(ee), —OC(═O)R^(ee), —OCO₂R^(ee), —C(═O)N(R^(ff))₂, —OC(═O)N(R^(ff))₂, —NR^(ff)C(═)R^(ee), —NR^(ff)CO₂R^(ee), —NR^(ff)C(═O)N(R^(ff))₂, —C(═NR^(ff))OR^(ee), —OC(═NR^(ff))R^(ee), —OC(═NR^(ff))OR^(ee), —C(═NR^(ff))N(R^(ff))₂, —OC(═NR^(ff))N(R^(ff))₂, —NR^(ff)C(═NR^(ff))N(R^(ff))₂, —NR^(ff)SO₂R^(ee), —SO₂N(R^(ff))₂, —SO₂R^(ee), —SO₂OR^(ee), —OSO₂R^(ee), —S(═O)R^(ee), —Si(R^(ee))₃, —OSi(R^(ee))₃, —C(═S)N(R^(ff))₂, —C(═O)SR^(ee), —C(═S)SR^(ee), —SC(═S)SR^(ee), —P(═O)₂R^(ee), —P(═O)(R^(ee))₂, —OP(═O)(R^(ee))₂, —OP(═O)(OR^(ee))₂, C₁₋₆ alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, heteroC₁₋₆alkyl, heteroC₂₋₆alkenyl, heteroC₂₋₆alkynyl, C₃₋₁₀ carbocyclyl, 3-10 membered heterocyclyl, C₆₋₁₀ aryl, 5-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups, or two geminal R^(dd) substituents can be joined to form ═O or ═S;

each instance of R^(ee) is, independently, selected from C₁₋₆ alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, heteroC₁₋₆ alkyl, heteroC₂₋₆alkenyl, heteroC₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl, C₆₋₁₀ aryl, 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups;

each instance of R^(ff) is, independently, selected from hydrogen, C₁₋₆ alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, heteroC₁₋₆alkyl, heteroC₂₋₆alkenyl, heteroC₂₋₆alkynyl, C₃₋₁₀ carbocyclyl, 3-10 membered heterocyclyl, C₆₋₁₀ aryl and 5-10 membered heteroaryl, or two R^(ff) groups are joined to form a 3-10 membered heterocyclyl or 5-10 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups; and

each instance of R^(gg) is, independently, halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OC₁₋₆ alkyl, —ON(C₁₋₆ alkyl)₂, —N(C₁₋₆ alkyl)₂, —N(C₁₋₆ alkyl)₃ ⁺X⁻, —NH(C₁₋₆ alkyl)₂ ⁺X⁻, —NH₂(C₁₋₆ alkyl)⁺X⁻, —NH₃ ⁺X⁻, —N(OC₁₋₆ alkyl)(C₁₋₆ alkyl), —N(OH)(C₁₋₆ alkyl), —NH(OH), —SH, —SC₁₋₆ alkyl, —SS(C₁₋₆ alkyl), —C(═O)(C₁₋₆ alkyl), —CO₂H, —CO₂(C₁₋₆ alkyl), —OC(═O)(C₁₋₆ alkyl), —OCO₂(C₁₋₆ alkyl), —C(═O)NH₂, —C(═O)N(C₁₋₆ alkyl)₂, —OC(═O)NH(C₁₋₆ alkyl), —NHC(═O)(C₁₋₆ alkyl), —N(C₁₋₆ alkyl)C(═O)(C₁₋₆ alkyl), —NHCO₂(C₁₋₆ alkyl), —NHC(═O)N(C₁₋₆ alkyl)₂, —NHC(═O)NH(C₁₋₆ alkyl), —NHC(═O)NH₂, —C(═NH)O(C₁₋₆ alkyl), —OC(═NH)(C₁₋₆ alkyl), —OC(═NH)OC₁₋₆ alkyl, —C(═NH)N(C₁₋₆ alkyl)₂, —C(═NH)NH(C₁₋₆ alkyl), —C(═NH)NH₂, —OC(═NH)N(C₁₋₆ alkyl)₂, —OC(NH)NH(C₁₋₆ alkyl), —OC(NH)NH₂, —NHC(NH)N(C₁₋₆ alkyl)₂, —NHC(═NH)NH₂, —NHSO₂(C₁₋₆ alkyl), —SO₂N(C₁₋₆ alkyl)₂, —SO₂NH(C₁₋₆alkyl), —SO₂NH₂, —SO₂C₁₋₆ alkyl, —SO₂OC₁₋₆ alkyl, —OSO₂C₁₋₆ alkyl, —SOC₁₋₆ alkyl, —Si(C₁₋₆ alkyl)₃, —OSi(C₁₋₆ alkyl)₃-C(═S)N(C₁₋₆ alkyl)₂, C(═S)NH(C₁₋₆ alkyl), C(═S)NH₂, —C(═O)S(C₁₋₆ alkyl), —C(═S)SC₁₋₆ alkyl, —SC(═S)SC₁₋₆ alkyl, —P(═O)₂(C₁₋₆ alkyl), —P(═O)(C₁₋₆ alkyl)₂, —OP(═O)(C₁₋₆ alkyl)₂, —OP(═O)(OC₁₋₆ alkyl)₂, C₁₋₆ alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, heteroC₁₋₆alkyl, heteroC₂₋₆alkenyl, heteroC₂₋₆alkynyl, C₃₋₁₀ carbocyclyl, C₆₋₁₀ aryl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl; or two geminal R^(gg) substituents can be joined to form ═O or ═S; wherein X⁻ is a counterion.

In certain embodiments, the carbon atom substituents are independently halogen, substituted or unsubstituted C₁₋₆ alkyl, —OR^(aa), —SR^(aa), —N(R^(bb))₂, —CN, —SCN, —NO₂, —C(═O)R^(aa), —CO₂R^(aa), —C(═O)N(R^(bb))₂, —OC(═O)R^(aa), —OCO₂R^(aa), —OC(═O)N(R^(bb))₂, —NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa), or —NR^(bb)C(═O)N(R^(bb))₂. In certain embodiments, the carbon atom substituents are independently halogen, substituted or unsubstituted C₁₋₆ alkyl, —OR^(aa), —SR^(aa), —N(R^(bb))₂, —CN, —SCN, or —NO₂.

The term “halo” or “halogen” refers to fluorine (fluoro, —F), chlorine (chloro, —Cl), bromine (bromo, —Br), or iodine (iodo, —I).

The term “hydroxyl” or “hydroxy” refers to the group —OH. The term “substituted hydroxyl” or “substituted hydroxyl,” by extension, refers to a hydroxyl group wherein the oxygen atom directly attached to the parent molecule is substituted with a group other than hydrogen, and includes groups selected from —OR^(aa), —ON(R^(bb))₂, —OC(═O)SR^(aa), —OC(═O)R^(aa), —OCO₂R^(aa), —OC(═O)N(R^(bb))₂, —OC(═NR^(bb))R^(aa), —OC(═NR^(bb))OR^(aa), —OC(═NR^(bb))N(R^(bb))₂, —OS(═O)R^(aa), —OSO₂R^(aa), —OSi(R^(aa))₃, —OP(R^(cc))₂, —OP(R^(cc))₃, —OP(═O)₂R^(aa), —OP(═O)(R^(aa))₂, —OP(═O)(OR^(cc))₂, —OP(═O)₂N(R^(bb))₂, and —OP(═O)(NR^(bb))₂, wherein R^(aa), R^(bb), and R^(cc) are as defined herein.

The term “thiol” or “thio” refers to the group —SH. The term “substituted thiol” or “substituted thio,” by extension, refers to a thiol group wherein the sulfur atom directly attached to the parent molecule is substituted with a group other than hydrogen, and includes groups selected from —SR^(aa), —S═SR^(cc), —SC(═S)SR^(aa), —SC(═O)SR^(aa), —SC(═O)OR^(aa), and —SC(═O)R^(aa), wherein R^(aa) and R^(cc) are as defined herein.

The term “amino” refers to the group —NH₂. The term “substituted amino,” by extension, refers to a monosubstituted amino, a disubstituted amino, or a trisubstituted amino. In certain embodiments, the “substituted amino” is a monosubstituted amino or a disubstituted amino group.

The term “monosubstituted amino” refers to an amino group wherein the nitrogen atom directly attached to the parent molecule is substituted with one hydrogen and one group other than hydrogen, and includes groups selected from —NH(R^(bb)), —NHC(═O)R^(aa), —NHCO₂R^(aa), —NHC(═O)N(R^(bb))₂, —NHC(═NR^(bb))N(R^(bb))₂, —NHSO₂R^(aa), —NHP(═O)(OR^(cc))₂, and —NHP(═O)(NR^(bb))₂, wherein R^(aa), R^(bb) and R^(cc) are as defined herein, and wherein R^(bb) of the group —NH(R^(bb)) is not hydrogen.

The term “disubstituted amino” refers to an amino group wherein the nitrogen atom directly attached to the parent molecule is substituted with two groups other than hydrogen, and includes groups selected from —N(R^(bb))₂, —NR^(bb) C(═O)R^(aa), —NR^(bb)CO₂R^(aa), —NR^(bb)C(═O)N(R^(bb))₂, —NR^(bb)C(═NR^(bb))N(R^(bb))₂, —NR^(bb)SO₂R^(aa), —NR^(bb)P(═O)(OR^(cc))₂, and —NR^(bb)P(═O)(NR^(bb))₂, wherein R^(aa), R^(bb), and R^(cc) are as defined herein, with the proviso that the nitrogen atom directly attached to the parent molecule is not substituted with hydrogen.

The term “trisubstituted amino” refers to an amino group wherein the nitrogen atom directly attached to the parent molecule is substituted with three groups, and includes groups selected from —N(R^(bb))₃ and —N(R^(bb))₃ ⁺X⁻, wherein R^(bb) and X⁻ are as defined herein.

The term “sulfonyl” refers to a group selected from —SO₂N(R^(bb))₂, —SO₂R^(aa), and —SO₂OR^(aa), wherein R^(aa) and R^(bb) are as defined herein.

The term “sulfinyl” refers to the group —S(═O)R^(aa), wherein R^(aa) is as defined herein.

The term “carbonyl” refers a group wherein the carbon directly attached to the parent molecule is sp² hybridized, and is substituted with an oxygen, nitrogen or sulfur atom, e.g., a group selected from ketones (—C(═O)R^(aa)), carboxylic acids (—CO₂H), aldehydes (—CHO), esters (—CO₂R^(aa), —C(═O)SR^(aa), —C(═S)SR^(aa)), amides (—C(═O)N(R^(bb))₂, —C(═O)NR^(bb)SO₂R^(aa), —C(═S)N(R^(bb))₂), and imines (—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa)), —C(═NR^(bb))N(R^(bb))₂), wherein R^(aa) and R^(bb) are as defined herein.

The term “silyl” refers to the group —Si(R^(aa))₃, wherein R^(aa) is as defined herein.

The term “boronyl” refers to boranes, boronic acids, boronic esters, borinic acids, and borinic esters, e.g., boronyl groups of the formula —B(R^(aa))₂, —B(OR^(cc))₂, and —BR^(aa)(OR^(cc)), wherein R^(aa) and R^(cc) are as defined herein.

The term “phosphino” refers to the group —P(R^(cc))₃, wherein R^(cc) is as defined herein. An exemplary phosphino group is triphenylphosphine.

The term “phosphono” refers to the group —O(P═O)(OR^(cc))R^(aa), wherein R^(aa) and R^(cc) are as defined herein.

The term “phosphoramido” refers to the group —O(P═O)(NR^(bb))₂, wherein each R^(bb) is as defined herein.

The term “oxo” refers to the group ═O, and the term “thiooxo” refers to the group ═S.

Nitrogen atoms can be substituted or unsubstituted as valency permits, and include primary, secondary, tertiary, and quaternary nitrogen atoms. Exemplary nitrogen atom substituents include, but are not limited to, hydrogen, —OH, —OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa), —C(═O)N(R^(cc))₂, —CO₂R^(aa), —SO₂R^(aa), —C(═NR^(bb))R^(aa), —C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc), —SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc), —P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂, —P(═O)(NR^(cc))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroC₁₋₁₀alkyl, heteroC₂₋₁₀alkenyl, heteroC₂₋₁₀alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(cc) groups attached to an N atom are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups, and wherein R^(aa), R^(bb), R^(cc) and R^(dd) are as defined above.

In certain embodiments, the substituent present on the nitrogen atom is an nitrogen protecting group (also referred to herein as an “amino protecting group”). Nitrogen protecting groups include, but are not limited to, —OH, —OR^(aa), —N(R^(cc))₂, —C(═O)R^(aa), —C(═O)N(R^(cc))₂, —CO₂R^(aa), —SO₂R^(aa), —C(═NR^(cc))R^(aa), —C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc), —SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc), C₁₋₁₀ alkyl (e.g., aralkyl, heteroaralkyl), C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroC₁₋₁₀ alkyl, heteroC₂₋₁₀ alkenyl, heteroC₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl groups, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups, and wherein R^(aa), R^(bb), R^(cc) and R^(dd) are as defined herein. Nitrogen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley & Sons, 1999, incorporated herein by reference.

For example, nitrogen protecting groups such as amide groups (e.g., —C(═O)R^(aa)) include, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N′-dithiobenzyloxyacylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenyl azophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide and o-(benzoyloxymethyl)benzamide.

Nitrogen protecting groups such as carbamate groups (e.g., —C(═O)OR^(aa)) include, but are not limited to, methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and 4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC or Boc), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzyl carbamate.

Nitrogen protecting groups such as sulfonamide groups (e.g., —S(═O)₂R^(aa)) include, but are not limited to, p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), 0-trimethyl silylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Other nitrogen protecting groups include, but are not limited to, phenothiazinyl-(10)-acyl derivative, N′-p-toluenesulfonylaminoacyl derivative, N′-phenylaminothioacyl derivative, N-benzoylphenylalanyl derivative, N-acetylmethionine derivative, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethyl silyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methyl amine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, N-p-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N—(N,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivative, N-diphenylborinic acid derivative, N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).

In certain embodiments, the substituent present on an oxygen atom is an oxygen protecting group (also referred to herein as an “hydroxyl protecting group”). Oxygen protecting groups include, but are not limited to, —R^(aa), —N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa), —CO₂R^(aa), —C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂, —S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃, —P(R^(cc))₂, —P(R^(cc))₃, —P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and —P(═O)(NR^(bb))₂, wherein R^(aa), R^(bb), and R^(cc) are as defined herein. Oxygen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley & Sons, 1999, incorporated herein by reference.

Exemplary oxygen protecting groups include, but are not limited to, methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethyl silyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethyl silylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl, 4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4′,4″-tris(levulinoyloxyphenyl)methyl, 4,4′,4″-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc), isobutyl carbonate, vinyl carbonate, allyl carbonate, t-butyl carbonate (BOC or Boc), p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzyl carbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzyl carbonate, p-nitrobenzyl carbonate, S-benzyl thiocarbonate, 4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts).

In certain embodiments, the substituent present on a sulfur atom is a sulfur protecting group (also referred to as a “thiol protecting group”). Sulfur protecting groups include, but are not limited to, —R^(aa), —N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa), —CO₂R^(aa), —C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂, —S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃, —P(R^(cc))₂, —P(R^(cc))₃, —P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and —P(═O)(NR^(bb))₂, wherein R^(aa), R^(bb), and R^(cc) are as defined herein. Sulfur protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley & Sons, 1999, incorporated herein by reference.

The term “heteroatom” refers to an atom that is not hydrogen or carbon. In certain embodiments, the heteroatom is a nitrogen, oxygen, or sulfur. In certain embodiments, the heteroatom is a nitrogen or oxygen. In certain embodiments, the heteroatom is nitrogen. In certain embodiments, the heteroatom is oxygen. In certain embodiments, the heteroatom is sulfur.

A “counterion” or “anionic counterion” is a negatively charged group associated with a positively charged group in order to maintain electronic neutrality. An anionic counterion may be monovalent (i.e., including one formal negative charge). An anionic counterion may also be multivalent (i.e., including more than one formal negative charge), such as divalent or trivalent. Exemplary counterions include halide ions (e.g., F⁻, Cl⁻, Br⁻, I⁻), NO₃ ⁻, ClO₄ ⁻, OH⁻, H₂PO₄ ⁻, HCO₃ ⁻, HSO₄ ⁻, sulfonate ions (e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate, naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonic acid-2-sulfonate, and the like), carboxylate ions (e.g., acetate, propanoate, benzoate, glycerate, lactate, tartrate, glycolate, gluconate, and the like), BF₄ ⁻, PF₄ ⁻, PF₆ ⁻, AsF₆ ⁻, SbF₆ ⁻, B[3,5-(CF₃)₂C₆H₃]₄]⁻, B(C₆F₅)₄ ⁻, BPh₄ ⁻, Al(OC(CF₃)₃)₄ ⁻, and carborane anions (e.g., CB₁₁H₁₂ ⁻ or (HCB₁₁Me₅Br₆)⁻). Exemplary counterions which may be multivalent include CO₃ ²⁻, HPO₄ ²⁻, PO₄ ³⁻, B₄O₇ ²⁻, SO₄ ²⁻, S₂O₃ ²⁻, carboxylate anions (e.g., tartrate, citrate, fumarate, maleate, malate, malonate, gluconate, succinate, glutarate, adipate, pimelate, suberate, azelate, sebacate, salicylate, phthalates, aspartate, glutamate, and the like), and carboranes.

The term “solvate” refers to forms of the compound, or a salt thereof, that are associated with a solvent, usually by a solvolysis reaction. This physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. The compounds described herein may be prepared, e.g., in crystalline form, and may be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include both stoichiometric solvates and non-stoichiometric solvates. In certain instances, the solvate will be capable of isolation, for example, when one or more solvent molecules are incorporated in the crystal lattice of a crystalline solid. “Solvate” encompasses both solution-phase and isolatable solvates. Representative solvates include hydrates, ethanolates, and methanolates.

The term “tautomers” or “tautomeric” refers to two or more interconvertible compounds resulting from at least one formal migration of a hydrogen atom and at least one change in valency (e.g., a single bond to a double bond, a triple bond to a single bond, or vice versa). The exact ratio of the tautomers depends on several factors, including temperature, solvent, and pH. Tautomerizations (i.e., the reaction providing a tautomeric pair) may catalyzed by acid or base. Exemplary tautomerizations include keto-to-enol, amide-to-imide, lactam-to-lactim, enamine-to-imine, and enamine-to-(a different enamine) tautomerizations.

It is also to be understood that compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed “isomers”. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers”.

Stereoisomers that are not mirror images of one another are termed “diastereomers” and those that are non-superimposable mirror images of each other are termed “enantiomers”. When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) or (−)-isomers respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a “racemic mixture”.

The term “polymer” refers to a molecule including two or more (e.g., 3 or more, 4 or more, 5 or more, 10 or more) repeating units which are covalently bound together. In certain embodiments, a polymer comprises 3 or more, 5 or more, 10 or more, 50 or more, 100 or more, 500 or more, 1000 or more, 2000 or more, 3000 or more, 4000 or more, 5000 or more, 6000 or more, 7000 or more, 8000 or more, 9000 or more, or 10000 or more repeating units. In certain embodiments, a polymer comprises more than 5000 repeating units. The repeating units of a polymer are referred to as “monomers.” A “homopolymer” is a polymer that consists of a single repeating monomer. A “copolymer” is a polymer that comprises two or more different monomer subunits. Copolymers include, but are not limited to, random, block, alternating, segmented, linear, branched, grafted, and tapered copolymers. A “graft polymer” is a segmented copolymer with a linear backbone of one composite and randomly distributed branches of another composite. The major difference between graft polymers and bottlebrush polymers (or brush-arm polymers) is the grafting density. The targeted graft density for bottlebrush polymers is that in at least one segment of the copolymer is one graft from each backbone monomer unit. A “star polymer” is a polymer that consists of several polymers chains connected at a core atom, core molecule, or core polymer. Polymers may be natural (e.g., naturally occurring polypeptides), or synthetic (e.g., non-naturally occurring). A polymer may have an overall molecular weight of 50 Da or greater, 100 Da or greater, 500 Da or greater, 1000 Da or greater, 2000 Da or greater, 5000 Da or greater, 10000 Da or greater, 20000 Da or greater, or 50000 Da or greater. Exemplary polymers include, without limitation, poly(ethylene glycol) 200 (PEG200), PEG400, PEG600, PEG800, PEG1000, PEG1500, PEG2000, PEG3000, PEG4000, and PEG6000.

The terms “living polymer” and “living polymerization” refer a polymerization where the ability of a growing polymer chain to terminate has been removed. Chain termination and chain transfer reactions are absent, and the rate of the chain initiation is also much larger than the rate of chain propagation. The result is that the polymer chains grow at a constant rate than see in traditional chain polymerization and their lengths remain very similar.

The terms “number average molecular weight,” “number average molar mass,” and “M_(n)” are measurements of the molecular mass of a polymer. The number average molecular mass is the ordinary arithmetic mean or average of the molecular masses of the individual polymers. It is determined by measuring the molecular mass of n polymer molecules, summing the masses, and dividing by n. For example, a polymer having 100 repeating units of a monomer with a molecular weight of 100 g/mol would have a number average molecular weight (M_(n)) of 10,000 g/mol [M_(n)=(100)*(100 g/mol)/(1)=10,000 g/mol)]. The number average molecular mass of a polymer can be determined by gel permeation chromatography, viscometry via the Mark-Houwink equation, colligative methods such as vapor pressure osmometry, end-group determination, or ¹H NMR.

The term “monomer” refers to a molecule that may bind covalently to other molecules to form a polymer. The process by which the monomers are combined to form a polymer is called polymerization. A macromolecule with one end-group that enables it to act as a monomer is called a macromonomer. Molecules made of a small number of monomer units are called oligomers.

The term “solvent” refers to a substance that dissolves one or more solutes, resulting in a solution. A solvent may serve as a medium for any reaction or transformation described herein. The solvent may dissolve one or more reactants or reagents in a reaction mixture. The solvent may facilitate the mixing of one or more reagents or reactants in a reaction mixture. The solvent may also serve to increase or decrease the rate of a reaction relative to the reaction in a different solvent. Solvents can be polar or non-polar, protic or aprotic. Common organic solvents useful in the methods described herein include, but are not limited to, acetone, acetonitrile, benzene, benzonitrile, 1-butanol, 2-butanone, butyl acetate, tert-butyl methyl ether, carbon disulfide carbon tetrachloride, chlorobenzene, 1-chlorobutane, chloroform, cyclohexane, cyclopentane, 1,2-dichlorobenzene, 1,2-dichloroethane, dichloromethane (DCM), N,N-dimethylacetamide N,N-dimethylformamide (DMF), 1,3-dimethyl-3,4,5,6-tetrahydro-2-pyrimidinone (DMPU), 1,4-dioxane, 1,3-dioxane, diethylether, 2-ethoxyethyl ether, ethyl acetate, ethyl alcohol, ethylene glycol, dimethyl ether, heptane, n-hexane, hexanes, hexamethylphosphoramide (HMPA), 2-methoxyethanol, 2-methoxyethyl acetate, methyl alcohol, 2-methylbutane, 4-methyl-2-pentanone, 2-methyl-1-propanol, 2-methyl-2-propanol, 1-methyl-2-pyrrolidinone, dimethylsulfoxide (DMSO), nitromethane, 1-octanol, pentane, 3-pentanone, 1-propanol, 2-propanol, pyridine, tetrachloroethylene, tetrahyrdofuran (THF), 2-methyltetrahydrofuran, toluene, trichlorobenzene, 1,1,2-trichlorotrifluoroethane, 2,2,4-trimethylpentane, trimethylamine, triethylamine, N,N-diisopropylethylamine, diisopropylamine, water, o-xylene, p-xylene.

A “subject” to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) and/or other non-human animals, for example, mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and/or dogs); and birds (e.g., commercially relevant birds such as chickens, ducks, geese, and/or turkeys). In certain embodiments, the animal is a mammal. In certain embodiments, the animal is a mouse. In certain embodiments, the animal is a human. The animal may be a male or female at any stage of development. The animal may be a transgenic animal or genetically engineered animal. In certain embodiments, the subject is a non-human animal.

The term “administer,” “administering,” or “administration” refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a compound or cell described herein or generated as described herein, or a composition thereof, in or on a subject.

As used herein, the term “salt” refers to any and all salts, and encompasses pharmaceutically acceptable salts.

As used herein, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N⁺(C₁₋₄ alkyl)₄ ⁻ salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

An “effective amount” of a compound described herein refers to an amount sufficient to elicit the desired biological response, i.e., treating the condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a compound described herein may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject. An effective amount encompasses therapeutic and prophylactic treatment.

A “therapeutically effective amount” of a compound described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the condition, and/or enhances the therapeutic efficacy of another therapeutic agent.

The term “small molecule” refers to molecules, whether naturally-occurring or artificially created (e.g., via chemical synthesis) that have a relatively low molecular weight. Typically, a small molecule is an organic compound (i.e., it contains carbon). The small molecule may contain multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxyl, carbonyls, and heterocyclic rings, etc.). In certain embodiments, the molecular weight of a small molecule is not more than about 1,000 g/mol, not more than about 900 g/mol, not more than about 800 g/mol, not more than about 700 g/mol, not more than about 600 g/mol, not more than about 500 g/mol, not more than about 400 g/mol, not more than about 300 g/mol, not more than about 200 g/mol, or not more than about 100 g/mol. In certain embodiments, the molecular weight of a small molecule is at least about 100 g/mol, at least about 200 g/mol, at least about 300 g/mol, at least about 400 g/mol, at least about 500 g/mol, at least about 600 g/mol, at least about 700 g/mol, at least about 800 g/mol, or at least about 900 g/mol, or at least about 1,000 g/mol. Combinations of the above ranges (e.g., at least about 200 g/mol and not more than about 500 g/mol) are also possible. In certain embodiments, the small molecule is a therapeutically active agent such as a drug (e.g., a molecule approved by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (C.F.R.)). The small molecule may also be complexed with one or more metal atoms and/or metal ions. In this instance, the small molecule is also referred to as a “small organometallic molecule.” Preferred small molecules are biologically active in that they produce a biological effect in animals, preferably mammals, more preferably humans. Small molecules include, but are not limited to, radionuclides and imaging agents.

The terms “imaging agent” and “contrast agent” refer to a substance used to enhance the contrast of structures or fluids within the body in medical imaging. It is commonly used to enhance the visibility of blood vessels and the gastrointestinal tract in medical imaging.

The term “crosslinker” refers to a compound that allows for two or more molecules or polymers to be joined by covalent bonds. In certain embodiments, the crosslinker results in a covalent attachment between two polymers.

The term “ring-opening metathesis polymerization (ROMP)” refers to a type of olefin metathesis chain-growth polymerization that is driven by the relief of ring strain in cyclic olefins (e.g. norbornene or cyclopentene). The catalysts used in the ROMP reaction include RuCl₃/alcohol mixture, bis(cyclopentadienyl)dimethylzirconium(IV), dichloro[1,3-bis(2,6-isopropylphenyl)-2-imidazolidinylidene](benzylidene)(tricyclohexylphosphine)ruthenium(II), dichloro[1,3-Bis(2-methylphenyl)-2-imidazolidinylidene](benzylidene)(tricyclohexylphosphine) ruthenium(II), dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene][3-(2-pyridinyl)propylidene]ruthenium(II), dichloro(3-methyl-2-butenylidene)bis (tricyclopentylphosphine)ruthenium(II), dichloro[1,3-bis(2-methylphenyl)-2-imidazolidinylidene](2-isopropoxyphenylmethylene)ruthenium(II) (Grubbs C571), dichloro(benzylidene)bis(tricyclohexylphosphine)ruthenium(II) (Grubbs I), dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene](benzylidene)(tricyclohexylphosphine) ruthenium(II) (Grubbs II), and dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene](benzylidene)bis(3-bromopyridine)ruthenium(II) (Grubbs III).

The terms “electron paramagnetic resonance (EPR) spectroscopy” or “electron spin resonance (ESR) spectroscopy” refer to a method for studying materials with unpaired electrons. EPR relies on the excitation of electron spins and is particularly useful for studying metal complexes or organic radicals (e.g., nitroxide radicals).

The term “imaging” refers to a technique and process of creating visual representations of the interior of a body or portion thereof (e.g., brain, heart, lung, liver, kidney, spleen, muscle, tissue, and tumor) for clinical analysis and medical intervention, as well as visualization of the function of organs and/or tissues. Medical imaging seeks to reveal internal structures hidden by the skin and bones, as well as to diagnose and sometimes treat disease. Medical imaging also establishes a database of normal anatomy and physiology to make it possible to identify abnormalities. Examples of imaging modalities include, but are not limited to, radiography, magnetic resonance imaging (MRI), nuclear medicine, ultrasound, elastography, tactile imaging, photoacoustic imaging, tomography, echocardiography, near-infrared fluorescence (NIRF) imaging, and magnetic particle imaging.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which constitute a part of this specification, illustrate several exemplary embodiments of the invention and together with the description, serve to explain certain principles of the invention. The embodiments disclosed in the drawings are exemplary and do not limit the scope of this disclosure.

FIGS. 1A-1B show chemical structures of BASP components (FIG. 1A) and the general brush-first ROMP procedure (FIG. 1B). Branched MMs chex-MM and Cy-MM are combined in the ratio j:0.01j. This combination of MMs is exposed to 1.0 equivalents of Grubbs III initiator to produce a living bottlebrush with an average degree of polymerization (DP)=j+0.01j=m. N equivalents of Acetal-XL is then added (in aliquots of 5 eq. Acetal-XL every 5 minutes) to provide the final BASP-ORCA. The properties of the BASP-ORCAs are defined by their m and N values (see Table 1).

FIGS. 2A-2C show the transmission electron microscopy image of BASP-ORCA1 (D_(TEM)=37±7) after being negatively stained with uranyl acetate (FIG. 2A), electron paramagnetic resonance (EPR) spectra for BASP-ORCA1 and chex-MM (FIG. 2B), and T₁ and T₂-weighted MRI phantoms for BASP-ORCA1, chex-MM, PBS buffer, chex-bottlebrush, and a PEG-BASP lacking chex (FIG. 2C). In FIG. 2C the concentration of chex-containing samples (BASP-ORCA1, chex-MM, and chex-bottlebrush) ranges from 1 mM to 4 mM chex. The concentration of PEG-BASP lacking chex ranges from 6 mg/mL to 21 mg/mL, which is equivalent to the mass per volume concentration range of BASP-ORCA1.

FIGS. 3A-3C are EPR spectra for BASP-ORCA1 at 1 minute, 40 minutes, and 180 minutes following exposure to 20 equivalents of sodium ascorbate (Asc) per nitroxide moeity (FIG. 3A), ascorbate reduction kinetics for BASP-ORCA1, chex-bottlebrush, and chex-MM (FIG. 3B), and Cy5.5 emission at 700 nm in response to Asc and glutathione (GSH) (FIG. 3C).

FIGS. 4A-4B show in vivo NIRF images of NCR nude mouse before and 20 hours after injection of BASP-ORCA1 (FIG. 4A) and ex vivo NIRF images of selected organs (FIG. 4B). Units of radiant efficiency:

$\frac{p\text{/}\sec \text{/}{cm}^{2}\text{/}{sr}}{\mu \; W\text{/}{cm}^{2}}.$

FIGS. 5A-5D show in vivo MRI imaging with BASP-ORCA. FIG. 5A shows T₂-weighted MR images of tumor bearing NCR nude mouse before (top row) and 20 hours after (bottom row) injection of 0.16 mmol chex/kg (“low dose”) of BASP-ORCA1. Each series of images corresponds to progressive slices in the z-axis through the tumor of the same mouse.

FIG. 5B shows T₂-weighted MR images of tumor bearing NCR nude mouse before (top row) and 20 hours after (bottom row) injection of 0.23 mmol chex/kg (“high dose”) of BASP-ORCA1. Each series of images corresponds to progressive slices in the z-axis through the tumor of the same mouse. FIG. 5C shows T₂-weighted coronal MR images before (top) and 20 hours after (bottom) injection of 0.23 mmol chex/kg (“high dose”) of BASP-ORCA1. FIG. 5D shows the percent MRI contrast change at various times following BASP-ORCA1 injection compared to pre-injection.

FIGS. 6A-6B show EPR spectra obtained for homogenized tissue samples collected for the same mice imaged in FIGS. 5A-5D 22 hours following BASP-ORCA1 injection (FIG. 6A) and fluorescence radiant efficiencies of the homogenized tissue samples (FIG. 6B). Right-hand bars: Muscle-normalized concentration of chex per milligram of protein as obtained from EPR integration of tissue homogenates. Left-hand bars: Muscle-normalized concentration of Cy5.5 in tissue homogenates as obtained from NIRF imaging.

FIGS. 7A-7B show GPC traces. FIG. 7A shows GPC traces of BASP-ORCAs with different brush length (m) and cross-linker equivalents (N). * indicates negligible residual MM; **denotes uncoupled bottlebrush. In all cases, the MM-to-bottlebrush conversions were almost quantitative, while the bottlebrush-to-BASP conversions were ≥85%. FIG. 7B shows GPC traces of chex-bottlebrush and PEG-BASP used for phantom MRI comparison with BASP-ORCA1.

FIG. 8 shows the EPR spectra of BASP-ORCAs of varying composition.

FIGS. 9A-9D show computational analysis of EPR spectra obtained during reduction kinetics experiments. t1=1 minutes, t16=40 minutes, t29=180 minutes following addition of Asc solution.

FIG. 10 shows the excitation and emission spectra of BASP-ORCA1.

FIG. 11 shows the cell viability assay for BASP-ORCA1 in the toxin-sensitive HUVEC and cancerous HeLa cell lines as measured by CellTiter Glo. No toxicity was observed until high concentrations were reached (up to 0.3 mg/mL and 5 mg/mL for HUVEC and HeLa, respectively).

FIG. 12 shows in vivo gross toxicity of BASP-ORCA1 following intravenous injections in BALB/c mice.

FIGS. 13A-13C show pharmacokinetics (PK) (FIG. 13A), biodistribution (BD) (FIG. 13B), and excrements collected 24 hours after administration of BASP-ORCA1 in BALB/c mice as imaged by NIRF (λ_(ex)/λ_(em)=640/700 nm) (FIG. 13C). PK data were fit into a two-component model using standard procedures (R²=0.95).^(101,102)

FIG. 14 shows ex vivo BD as assessed by NIRF imaging (λ_(ex)/λ_(em)=640/700 nm) of subcutaneous tumor-bearing NCR-NU mice following injection of BASP-ORCA1.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The present disclosure provides methods, compounds, particles, nanoparticles, compositions, systems, and kits focused on the synthesis and uses of brush-arm star polymers containing at least one imaging agent. In certain embodiments, the polymers are brush-arm star polymer organic radical contrast agents (BASP-ORCAs). In certain embodiments, the brush-arm star polymer organic radical contrast agents are comprised of brush-arm polymers covalently linked to a polymer core via crosslinkers. In certain embodiments, BASP-ORCAs contain a high concentration of reduction-resistant nitroxide groups bound between a poly(ethylene glycol) (PEG) shell and a polyacetal core.

These polymers are shown to be effective for medical imaging (e.g., brain, heart, lung, liver, kidney, spleen, muscle, tissue, and tumor). In certain embodiments, the imaging modality is magnetic resonance imaging. In certain embodiments, the imaging modality is near-infrared fluorescence imaging.

Brush-Arm Star Polymers

One aspect of the present disclosure relates to brush-arm star polymers comprising at least 100 repeating units selected from Formula (I) and Formula (II):

or a salt thereof, wherein:

each of A, A¹, and B is independently C₁-C₁₂ alkylene, C₂-C₁₂ alkenylene, C₂-C₁₂ alkynylene, or C₁-C₁₂ heteroalkylene, C₂-C₁₂ heteroalkenylene, C₂-C₁₂ heteroalkynylene, wherein each alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene is optionally substituted with 1-24 independently selected R¹;

X is an imaging agent;

P is alkylene, heteroalkylene, or polymer;

L is a bond, —O—, —S—, —S—S—, C₁-C₁₂ alkylene, C₂-C₁₂ alkenylene, C₂-C₁₂ alkynylene, C₁-C₁₂ heteroalkylene, (C₀-C₁₂ alkylene)-arylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-arylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ alkylene)-arylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ heteroalkylene)-arylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ alkylene)-heteroarylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heteroarylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heteroarylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ alkylene)-heterocyclylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heterocyclylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-aryl-(C₀-C₁₂ heteroalkylene), or (C₀-C₁₂ heteroalkylene)-heterocyclylene-(C₀-C₁₂ heteroalkylene), wherein each alkylene, alkenylene, alkynylene, heteroalkylene, arylene, heteroarylene, or heterocyclylene is optionally substituted with 1-24 independently selected R¹, and combinations thereof;

each R¹ is independently alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR^(A), —N(R^(A))₂, —NR^(A)C(O)R^(A), —NR^(A)C(O)OR^(A), —NR^(A)C(O)N(R^(A))₂, —C(O)N(R^(A))₂, —C(O)R^(A), —C(O)OR^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂, —SR^(A), or —S(O)_(m)R^(A);

each R^(A) is independently hydrogen, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, or C₁-C₆ haloalkyl; each of a and b are independently an integer between 1 and 10000, inclusive;

each of “1”, “2”, “3”, “4”, “5”, and “6” is independently a terminal group selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted acyl, optionally substituted hydroxyl, optionally substituted amino, and optionally substituted thio; or represents a bond to a structure of Formula (I) or Formula (II);

y is an integer between 1 and 100, inclusive; and

m is 1 or 2.

In certain embodiments, P is a polyether, polyester, polyacrylamide, polycarbonate, polysiloxane, polyfluorocarbon, polysulfone, or polystyrene. In certain embodiments, P is a polyether selected from the group consisting of polyethylene glycol (PEG), polyoxymethylene (POM), polypropylene glycol (PPG), polytetramethylene glycol (PTMG), poly(ethyl ethylene) phosphate (PEEP), and poly(oxazoline). In certain embodiments, P is a polyester selected from the group consisting of polyglycolic acid (PGA), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), polyhydroxyalkanoate (PHA), polyhydroxybutryate (PHB), polyethylene adipate (PEA), polybutylene succinate (PBS), or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). In certain embodiments, P is a poly(N-alkylacrylamide). In certain embodiments, P is a polycarbonate selected from the group consisting of poly(Bisphenol A carbonate), poly[Bisphenol A carbonate-co-4,4′-(3,3,5-trimethylcyclohexylidene)diphenol carbonate], or poly(propylene carbonate). In certain embodiments, P is a polysiloxane. In certain embodiments, P is polydimethylsiloxane (PDMS). In certain embodiments, P is a polyfluorocarbon selected from the group consisting of poly(chlorotrifluoroethylene), poly(ethylene-co-tetrafluoroethylene), poly(tetrafluoroethylene), poly(tetrafluoroethylene-co-perfluoro(propylvinyl ether)), poly(vinylidene fluoride), and poly(vinylidene fluoride-co-hexafluoropropylene). In certain embodiments, P is a polysulfone selected from the group consisting of poly[1-[4-(3-carboxy-4-hydroxyphenylazo)benzenesulfonamido]-1,2-ethanediyl, sodium salt], poly(1-hexadecene-sulfone), poly(oxy-1,4-phenylenesulfonyl-1,4-phenylene), poly(oxy-1,4-phenylenesulfonyl-1,4-phenylene), and polyphenylsulfone.

In certain embodiments, P is poly(ethylene glycol) with a molecular weight ranging from about 200 g/mol to about 6000 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 200 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 200 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 500 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 1000 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 1500 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 2000 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 2500 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 3000 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 3500 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 4000 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 4500 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 5000 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 5500 g/mol. In certain embodiments, P is poly(ethylene glycol) with a molecular weight about 6000 g/mol.

In certain embodiments, B is C₁-C₁₂ alkylene, optionally substituted with 1-24 independently selected R¹; R¹ is alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR^(A), —N(R^(A))₂, —NR^(A)C(O)R^(A), —NR^(A)C(O)OR^(A), —NR^(A)C(O)N(R^(A))₂, —C(O)N(R^(A))₂, —C(O)R^(A), —C(O)OR^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂, —SR^(A), or —S(O)_(m)R^(A); each R^(A) is independently hydrogen, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, or C₁-C₆ haloalkyl; and m is 1 or 2.

In certain embodiments, A is C₁-C₁₂ alkylene, optionally substituted with 1-24 independently selected R¹; R¹ is alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR^(A), —N(R^(A))₂, —NR^(A)C(O)R^(A), —NR^(A)C(O)OR^(A), —NR^(A)C(O)N(R^(A))₂, —C(O)N(R^(A))₂, —C(O)R^(A), —C(O)OR^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂, —SR^(A), or —S(O)_(m)R^(A); each R^(A) is independently hydrogen, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, or C₁-C₆ haloalkyl; and m is 1 or 2.

In certain embodiments, A¹ is C₁-C₁₂ alkylene, optionally substituted with 1-24 independently selected R¹; R¹ is alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR^(A), —N(R^(A))₂, —NR^(A)C(O)R^(A), —NR^(A)C(O)OR^(A), —NR^(A)C(O)N(R^(A))₂, —C(O)N(R^(A))₂, —C(O)R^(A), —C(O)OR^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂, —SR^(A), or —S(O)_(m)R^(A); each R^(A) is independently hydrogen, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, or C₁-C₆ haloalkyl; and m is 1 or 2.

In certain embodiments, L is selected from a group consisting of

wherein: q, p, and o are independently an integer between 0 and 20, inclusive.

In certain embodiments, L is independently selected from

In certain embodiments, X is a chelated metal, inorganic compound, organometallic compound, organic compound, or salt thereof. In certain embodiments, the imaging agent contains a metal selected from the group consisting of scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, mercury, rutherfordium, dubnium, seaborgium, bohrium, hassium, meitnerium, gadolinium, gallium, thallium, and barium. In certain embodiments, X is and inorganic compound. In certain embodiments, X is an organic compound. In certain embodiments, X is metal-free.

In certain embodiments, the imaging agent is an magnetic resonance imaging (MRI) agent. In certain embodiments, the MRI agent is a chelated gadolinium. In certain embodiments, the MRI agent is a nitroxide radical-containing compound.

In certain embodiments, the imaging agent is a nuclear medicine imaging agent. In certain embodiments, the nuclear medicine imaging agent is selected from the group consisting of ⁶⁴Cu diacetyl-bis(N⁴-methylthiosemicarbazone) (⁶⁴Cu-ASTM), ¹⁸F-fluorodeoxyglucose (FDG), ¹⁸F-fluoride, 3′-deoxy-3′-[¹⁸F]fluorothymi dine (FLT), and ¹⁸F-fluoromisonidazole (FMISO), chelated gallium, chelated technetium-99m, and chelated thallium.

In certain embodiments, the imaging agent is radiographic imaging agent. In certain embodiments, the radiographic imaging agent is selected from the group consisting of chelated barium, gastrografin, metrizoic acid, iotalamic acid, ioxaglate, iopamidol, iohexol, ioxilan, iopromide, iodixanol, and ioversol.

In certain embodiments, the imaging agent X is a radical-containing compound. In certain embodiments, the imaging agent is a nitroxide radical-containing compound. In certain embodiments, the imaging agent X is of the formula:

In certain embodiments, the imaging agent X is an organic compound. In certain embodiments, the imaging agent is a salt of an organic compound. In certain embodiments, the imaging agent X is of the formula:

In certain embodiments, the repeating unit of Formula (I) is of formula:

or a salt thereof, wherein:

each of A, A¹, and B is independently C₁-C₁₂ alkylene, C₂-C₁₂ alkenylene, C₂-C₁₂ alkynylene, or C₁-C₁₂ heteroalkylene, C₂-C₁₂ heteroalkenylene, C₂-C₁₂ heteroalkynylene, wherein each alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene is optionally substituted with 1-24 independently selected R¹;

X is an imaging agent;

P is alkylene, heteroalkylene, or polymer;

L is a bond, —O—, —S—, —S—S—, C₁-C₁₂ alkylene, C₂-C₁₂ alkenylene, C₂-C₁₂ alkynylene, C₁-C₁₂ heteroalkylene, (C₀-C₁₂ alkylene)-arylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-arylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ alkylene)-arylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ heteroalkylene)-arylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ alkylene)-heteroarylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heteroarylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heteroarylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ alkylene)-heterocyclylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heterocyclylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-aryl-(C₀-C₁₂ heteroalkylene), or (C₀-C₁₂ heteroalkylene)-heterocyclylene-(C₀-C₁₂ heteroalkylene), wherein each alkylene, alkenylene, alkynylene, heteroalkylene, arylene, heteroarylene, or heterocyclylene is optionally substituted with 1-24 independently selected R¹, and combinations thereof;

each R¹ is independently alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR^(A), —N(R^(A))₂, —NR^(A)C(O)R^(A), —NR^(A)C(O)OR^(A), —NR^(A)C(O)N(R^(A))₂, —C(O)N(R^(A))₂, —C(O)R^(A), —C(O)OR^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂, —SR^(A), or —S(O)_(m)R^(A);

each R^(A) is independently hydrogen, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, or C₁-C₆ haloalkyl;

y is an integer between 1 and 100, inclusive; and

m is 1 or 2.

In certain embodiments, the repeating unit is of formula:

In certain embodiments, the repeating unit is of the formula:

In certain embodiments, the repeating unit of Formula (II) is of formula:

or a salt thereof, wherein:

L is a bond, —O—, —S—, —S—S—, C₁-C₁₂ alkylene, C₂-C₁₂ alkenylene, C₂-C₁₂ alkynylene, C₁-C₁₂ heteroalkylene, (C₀-C₁₂ alkylene)-arylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-arylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ alkylene)-arylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ heteroalkylene)-arylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ alkylene)-heteroarylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heteroarylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heteroarylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ alkylene)-heterocyclylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heterocyclylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-aryl-(C₀-C₁₂ heteroalkylene), or (C₀-C₁₂ heteroalkylene)-heterocyclylene-(C₀-C₁₂ heteroalkylene), wherein each alkylene, alkenylene, alkynylene, heteroalkylene, arylene, heteroarylene, or heterocyclylene is optionally substituted with 1-24 independently selected R¹, and combinations thereof;

each R¹ is independently alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR^(A), —N(R^(A))₂, —NR^(A)C(O)R^(A), —NR^(A)C(O)OR^(A), —NR^(A)C(O)N(R^(A))₂, —C(O)N(R^(A))₂, —C(O)R^(A), —C(O)OR^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂, —SR^(A), or —S(O)_(m)R^(A);

each R^(A) is independently hydrogen, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, or C₁-C₆ haloalkyl; and

m is 1 or 2.

In certain embodiments, the repeating unit is of formula:

In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is between about 1:20 to about 20:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:20, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:19, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:18, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:17, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:16, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:15, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:14, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:13, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:12, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:11, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:10, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:9, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:8, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:7, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:6, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:5, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:4, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:3, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:2, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 1:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 2:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 3:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 4:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 5:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 6:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 7:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 8:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 9:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 10:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 11:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 12:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 13:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 14:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 15:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 16:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 17:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 18:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 19:1, respectively. In certain embodiments, the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is about 20:1, respectively.

In certain embodiments, the polymer forms a particle or nanoparticle of a diameter between about 10 nm and about 1000 nm. In certain embodiments, the polymer forms a particle of a diameter between about 10 nm and about 100 nm. In certain embodiments, the polymer forms a particle of a diameter between about 100 nm and about 200 nm. In certain embodiments, the polymer forms a particle of a diameter between about 200 nm and about 300 nm. In certain embodiments, the polymer forms a particle of a diameter between about 300 nm and about 400 nm. In certain embodiments, the polymer forms a particle of a diameter between about 400 nm and about 500 nm. In certain embodiments, the polymer forms a particle of a diameter between about 500 nm and about 600 nm. In certain embodiments, the polymer forms a particle of a diameter between about 600 nm and about 700 nm. In certain embodiments, the polymer forms a particle of a diameter between about 700 nm and about 800 nm. In certain embodiments, the polymer forms a particle of a diameter between about 800 nm and about 900 nm. In certain embodiments, the polymer forms a particle of a diameter between about 900 nm and about 1000 nm. In certain embodiments, the polymer forms a particle of a diameter between about 28 nm and about 49 nm. In certain embodiments, the polymer forms a particle of a diameter between about 25 nm and about 40 nm.

Methods for Preparing Brush-Arm Star Polymers

In another aspect of the present disclosure, a method of producing a brush-arm star polymer comprising an imaging agent is described herein, the method comprises the steps of: reacting one or more macromonomers containing an imaging agent with a metathesis catalyst to form a living polymer; and mixing a crosslinker with the living polymer. In certain embodiments, at least two different macromonomers each containing a different imaging agent are reacted.

In certain embodiments, the brush-arm star polymer is prepared by reacting macromonomer

macromonomer

a and ring-opening metathesis catalyst in a solvent to form a living polymer in the first step. In the second step the living polymer is then mixed with crosslinker

to form the brush-arm star polymer. In certain embodiments the ring-opening methathesis catalyst is Grubbs 3^(rd) generation bispyridyl catalyst (Grubbs III).

In certain embodiments, the reaction time of the first step is between about 10 minutes and about 60 minutes. In certain embodiments, the reaction time of the first step is about 30 minutes. In certain embodiments, the reaction time of the second step is between about 1 hour and about 24 hours. In certain embodiments, the reaction time of the second step is about 6 hours.

In certain embodiments, the solvent used to prepare the brush-arm star polymer can be polar or non-polar, protic or aprotic. Common organic solvents useful in the methods described herein include, but are not limited to, acetone, acetonitrile, benzene, benzonitrile, 1-butanol, 2-butanone, butyl acetate, tert-butyl methyl ether, carbon disulfide carbon tetrachloride, chlorobenzene, 1-chlorobutane, chloroform, cyclohexane, cyclopentane, 1,2-dichlorobenzene, 1,2-dichloroethane, dichloromethane (DCM), N,N-dimethylacetamide N,N-dimethylformamide (DMF), 1,3-dimethyl-3,4,5,6-tetrahydro-2-pyrimidinone (DMPU), 1,4-dioxane, 1,3-dioxane, diethylether, 2-ethoxyethyl ether, ethyl acetate, ethyl alcohol, ethylene glycol, dimethyl ether, heptane, n-hexane, hexanes, hexamethylphosphoramide (HMPA), 2-methoxyethanol, 2-methoxyethyl acetate, methyl alcohol, 2-methylbutane, 4-methyl-2-pentanone, 2-methyl-1-propanol, 2-methyl-2-propanol, 1-methyl-2-pyrrolidinone, dimethylsulfoxide (DMSO), nitromethane, 1-octanol, pentane, 3-pentanone, 1-propanol, 2-propanol, pyridine, tetrachloroethylene, tetrahyrdofuran (THF), 2-methyltetrahydrofuran, toluene, trichlorobenzene, 1,1,2-trichlorotrifluoroethane, 2,2,4-trimethylpentane, trimethylamine, triethylamine, N,N-diisopropylethylamine, diisopropylamine, water, o-xylene, p-xylene. In certain embodiments, the solvent used to prepare the brush-arm star polymer is tetrahyrdofuran (THF).

In certain embodiments, the molar ratio of chex-MM and Cy-MM is between about 1:1 and about 1000:1. In certain embodiments, the molar ratio of chex-MM and Cy-MM is about 100:1.

In certain embodiments, the molar ratio of (chex-MM+Cy-MM) and ring-opening metathesis catalyst is between about 1:1 and about 100:1. In certain embodiments, the molar ratio of (chex-MM+Cy-MM) and Grubbs (III) is about 5.05:1. In certain embodiments, the molar ratio of (chex-MM+Cy-MM) and Grubbs (III) is about 7.07:1. In certain embodiments, the molar ratio of (chex-MM+Cy-MM) and Grubbs (III) is about 9.99:1.

In certain embodiments, the molar equivalents of Acetal-XL with respect to Grubbs III is between about 1 equivalent and about 100 equivalents. In certain embodiments, the molar equivalents of Acetal-XL with respect to Grubbs III is about 15 equivalents. In certain embodiments, the molar equivalents of Acetal-XL with respect to Grubbs III is about 20 equivalents. In certain embodiments, the molar equivalents of Acetal-XL with respect to Grubbs III is about 30 equivalents.

In certain embodiments, the macromonomer is of Formula (III):

or a salt thereof, wherein: each of A, A¹, and B is independently C₁-C₁₂ alkylene, C₂-C₁₂ alkenylene, C₂-C₁₂ alkynylene, or C₁-C₁₂ heteroalkylene, C₂-C₁₂ heteroalkenylene, C₂-C₁₂ heteroalkynylene, wherein each alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene is optionally substituted with 1-24 independently selected R¹; X is an imaging agent; P is alkylene, heteroalkylene, or polymer; L is a bond, —O—, —S—, —S—S—, C₁-C₁₂ alkylene, C₂-C₁₂ alkenylene, C₂-C₁₂ alkynylene, C₁-C₁₂ heteroalkylene, (C₀-C₁₂ alkylene)-arylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-arylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ alkylene)-arylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ heteroalkylene)-arylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ alkylene)-heteroarylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heteroarylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heteroarylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ alkylene)-heterocyclylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heterocyclylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-aryl-(C₀-C₁₂ heteroalkylene), or (C₀-C₁₂ heteroalkylene)-heterocyclylene-(C₀-C₁₂ heteroalkylene), wherein each alkylene, alkenylene, alkynylene, heteroalkylene, arylene, heteroarylene, or heterocyclylene is optionally substituted with 1-24 independently selected R¹, and combinations thereof, each R¹ is independently alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR^(A), —N(R^(A))₂, —NR^(A)C(O)R^(A), —NR^(A)C(O)OR^(A), —NR^(A)C(O)N(R^(A))₂, —C(O)N(R^(A))₂, —C(O)R^(A), —C(O)OR^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂, —SR^(A), or —S(O)_(m)R^(A); each R^(A) is independently hydrogen, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, or C₁-C₆ haloalkyl; y is an integer between 1 and 100, inclusive; and m is 1 or 2.

In certain embodiments, P is poly(ethylene glycol) with a molecular weight ranging from about 200 g/mol to about 6000 g/mol.

In certain embodiments, B is C₁-C₁₂ alkylene, optionally substituted with 1-24 independently selected R¹; R¹ is alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR^(A), —N(R^(A))₂, —NR^(A)C(O)R^(A), —NR^(A)C(O)OR^(A), —NR^(A)C(O)N(R^(A))₂, —C(O)N(R^(A))₂, —C(O)R^(A), —C(O)OR^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂, —SR^(A), or —S(O)_(m)R^(A); each R^(A) is independently hydrogen, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, or C₁-C₆ haloalkyl; and m is 1 or 2.

In certain embodiments, A is C₁-C₁₂ alkylene, optionally substituted with 1-24 independently selected R¹; R¹ is alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR^(A), —N(R^(A))₂, —NR^(A)C(O)R^(A), —NR^(A)C(O)OR^(A), —NR^(A)C(O)N(R^(A))₂, —C(O)N(R^(A))₂, —C(O)R^(A), —C(O)OR^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂, —SR^(A), or —S(O)_(m)R^(A); each R^(A) is independently hydrogen, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, or C₁-C₆ haloalkyl; and m is 1 or 2.

In certain embodiments, A¹ is C₁-C₁₂ alkylene, optionally substituted with 1-24 independently selected R¹; R¹ is alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR^(A), —N(R^(A))₂, —NR^(A)C(O)R^(A), —NR^(A)C(O)OR^(A), —NR^(A)C(O)N(R^(A))₂, —C(O)N(R^(A))₂, —C(O)R^(A), —C(O)OR^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂, —SR^(A), or —S(O)_(m)R^(A); each R^(A) is independently hydrogen, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, or C₁-C₆ haloalkyl; and m is 1 or 2.

In certain embodiments, the metathesis catalyst is a ring-opening metathesis polymerization (ROMP) catalyst. In certain embodiments, the metathesis catalyst is a transition metal complex. In certain embodiments, the metal is selected from the group consisting of scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, mercury, rutherfordium, dubnium, seaborgium, bohrium, hassium, and meitnerium. In certain embodiments, the metathesis catalyst is a ruthenium complex. In certain embodiments, the metathesis catalyst is a molybdenum complex. In certain embodiments, the metathesis catalyst is a zirconium complex. In certain embodiments, the metathesis catalyst is selected from the group consisting of RuCl₃/alcohol mixture, bis(cyclopentadienyl)dimethylzirconium(IV), dichloro[1,3-bis(2,6-isopropylphenyl)-2-imidazolidinylidene](benzylidene)(tricyclohexylphosphine)ruthenium(II), dichloro[1,3-Bis(2-methylphenyl)-2-imidazolidinylidene](benzylidene)(tricyclohexylphosphine) ruthenium(II), dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene][3-(2-pyridinyl)propylidene]ruthenium(II), dichloro(3-methyl-2-butenylidene)bis (tricyclopentylphosphine)ruthenium(II), dichloro[1,3-bis(2-methylphenyl)-2-imidazolidinylidene](2-isopropoxyphenylmethylene)ruthenium(II) (Grubbs C571), dichloro(benzylidene)bis(tricyclohexylphosphine)ruthenium(II) (Grubbs I), dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene](benzylidene)(tricyclohexylphosphine) ruthenium(II) (Grubbs II), and dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene](benzylidene)bis(3-bromopyridine)ruthenium(II) (Grubbs III). In certain embodiment, the metathesis catalyst is of the formula:

Compositions and Kits

In one aspect of the present disclosure, compositions and kits are described herein. In certain embodiments, a composition is comprised of a polymer described herein and a pharmaceutically acceptable excipient. In certain embodiments, a composition is comprised of an effective amount of a polymer described herein.

Compositions described herein can be prepared by any method known in the art. In general, such preparatory methods include bringing the polymer described herein into association with a carrier or excipient, and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping, and/or packaging the product into a desired single- or multi-dose unit.

Compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. A “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition described herein will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. The composition may comprise between 0.1% and 100% (w/w) active ingredient.

Pharmaceutically acceptable excipients used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.

Although the descriptions of compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with ordinary experimentation.

The compounds and compositions provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via blood and/or lymph supply, and/or direct administration to an affected site. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and/or the condition of the subject (e.g., whether the subject is able to tolerate oral administration). In certain embodiments, the compound or pharmaceutical composition described herein is suitable for topical administration to the eye of a subject.

The exact amount of a compound required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound, mode of administration, and the like. An effective amount may be included in a single dose (e.g., single oral dose) or multiple doses (e.g., multiple oral doses). In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, any two doses of the multiple doses include different or substantially the same amounts of a compound or polymer described herein. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses a day, two doses a day, one dose a day, one dose every other day, one dose every third day, one dose every week, one dose every two weeks, one dose every three weeks, or one dose every four weeks. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is one dose per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is two doses per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses per day. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the duration between the first dose and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject, tissue, or cell. In certain embodiments, the duration between the first dose and last dose of the multiple doses is three months, six months, or one year. In certain embodiments, the duration between the first dose and last dose of the multiple doses is the lifetime of the subject, tissue, or cell.

Dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

The compound or composition can be administered concurrently with, prior to, or subsequent to one or more additional pharmaceutical agents, which may be useful as, e.g., combination therapies. Pharmaceutical agents include therapeutically active agents. Pharmaceutical agents also include prophylactically active agents. Pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved for human or veterinary use by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells. Each additional pharmaceutical agent may be administered at a dose and/or on a time schedule determined for that pharmaceutical agent. The additional pharmaceutical agents may also be administered together with each other and/or with the compound or composition described herein in a single dose or administered separately in different doses. The particular combination to employ in a regimen will take into account compatibility of the compound described herein with the additional pharmaceutical agent(s) and/or the desired therapeutic and/or prophylactic effect to be achieved. In general, it is expected that the additional pharmaceutical agent(s) in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.

Also encompassed by the disclosure are kits. The kits provided may comprise a pharmaceutical composition or compound described herein and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container). In some embodiments, provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a pharmaceutical composition or compound described herein. In some embodiments, the pharmaceutical composition or compound described herein provided in the first container and the second container are combined to form one unit dosage form.

In certain embodiments, the kits are comprised are comprised of a polymer described herein and instructions for use. In certain embodiments, the kits are comprised of a composition described herein and instructions for use.

In certain embodiments, a kit described herein further includes instructions for using the kit. A kit described herein may also include information as required by a regulatory agency such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kits is prescribing information. A kit described herein may include one or more additional pharmaceutical agents described herein as a separate composition.

Methods of Treatment

In one aspect of the present disclosure, methods of imaging a subject or a portion of a subject are described herein, the method comprising steps of: administering to a subject a polymer described herein, or a composition described herein; and acquiring an image. In certain embodiments, the imaging modality is selected from the group consisting of radiography, magnetic resonance imaging (MRI), nuclear medicine, ultrasound, elastography, tactile imaging, photoacoustic imaging, tomography, echocardiography, near-infrared fluorescence (NIRF) imaging, and magnetic particle imaging. In certain embodiments, the imaging modality is magnetic resonance imaging (MRI). In certain embodiments, the imaging modality is near-infrared fluorescence (NIRF) imaging.

In certain embodiments, the subject is an animal. The animal may be of either sex and may be at any stage of development. In certain embodiments, the subject described herein is a human. In certain embodiments, the subject is a non-human animal. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a non-human mammal. In certain embodiments, the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a companion animal, such as a dog or cat. In certain embodiments, the subject is a livestock animal, such as a cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a zoo animal. In some embodiments, the subject is a research animal, such as a rodent (e.g., mouse, rat), dog, pig, or non-human primate. In certain embodiments, the animal is a genetically engineered animal. In certain embodiments, the animal is a transgenic animal (e.g., transgenic mice and transgenic pigs).

In certain embodiments, the time period between administering to a subject a polymer described herein, or a composition described herein; and acquiring an image is between about 1 minute and about 100 hours. In certain embodiments, the time period between administering to a subject a polymer described herein, or a composition described herein; and acquiring an image is between about 1 hour and about 100 hours. In certain embodiments, the time period between administering to a subject a polymer described herein, or a composition described herein; and acquiring an image is between about 1 hour and about 50 hours. In certain embodiments, the time period between administering to a subject a polymer described herein, or a composition described herein; and acquiring an image is between about 1 hour and about 20 hours. In certain embodiments, the time period between administering to a subject a polymer described herein, or a composition described herein; and acquiring an image is between about 1 hour and about 10 hours. In certain embodiments, the time period between administering to a subject a polymer described herein, or a composition described herein; and acquiring an image is between about 1 hour and about 5 hours.

EXAMPLES

In order that the invention described herein may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the methods, compositions, and systems provided herein and are not to be construed in any way as limiting their scope.

BASP-ORCA Design and Synthesis

One of the most common ways to increase the relaxivity of MRI contrast agents (including nitroxides) involves attaching them to a rigid macromolecular scaffold. For example, Rajca et al., appended a spirocyclohexyl nitroxide derivative (“chex”)⁶⁹ to the surface of dendrimers to produce chex-dendrimer ORCAs where the per-chex r₁ was 0.42 mM⁻¹s⁻¹ compared to r₁=0.14 mM⁻¹s⁻¹ for the model nitroxide 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (3-CP). In anotherstudy, chex was appended to the core of PEGylated branched-bottlebrush polymers.⁷⁰ The resulting polymers had a per-chex r₁ of 0.32 mM⁻¹s⁻¹, which was approximately 50% greater than the chex-macromonomer used to synthesize these polymers (chex-MM, FIG. 1A). In this system, r₂ also increased from 0.30 mM⁻¹s⁻¹ for chex-MM to 0.82 mM⁻¹s⁻¹ for the chex-bottlebrush polymer, thus demonstrating that increasing the macromolecular size and chex density leads to increases in both r₁ and r₂, with a greater increase in r₂. In an effort to further increase these relaxivity values, the aim was to incorporate chex into the BASP macromolecules. Moreover, it was hypothesized that BASPs could provide enhanced nitroxide stability potentially making tumor imaging in vivo possible. The control and robustness of BASP synthesis would enable the scalable production of BASP-ORCAs with optimal sizes for tumor accumulation.

BASP-ORCAs were synthesized following the brush-first ring-opening metathesis polymerization (ROMP) strategy (FIGS. 1A-1B).^(71,72) Norbornene-based branched macromonomers (MMs, FIG. 1A) featuring 3 kDa (PEG) and either chex (chex-MM) or Cy5.5 dye (Cy-MM, FIG. 1A) were copolymerized by exposure to Grubbs 3^(rd) generation bis-pyridine initiator⁷³ (Grubbs III, FIG. 1A; reaction stoichiometry: j equivalents chex-MM: 0.01j Cy-MM:1.0 Grubbs III) for 30 minutes (FIG. 1B). The resulting living bottlebrush polymers with an average degree of polymerization (DP) of ˜j+0.01j=m were crosslinked via slow addition of N equivalents of bis-norbornene acetal crosslinker (Acetal-XL, FIG. 1A) directly to the reaction mixture to generate the desired BASP-ORCA (FIG. 1B). With this method, the BASP-ORCA size is determined by the MM:Grubbs III:Acetal-XL ratios (i.e., m and N values). Much less Cy-MM (0.01j) relative to chex-MM (j) was used to bridge the difference in concentration requirements between MRI (mM to μM) and NIRF (nM to pM).

To identify optimal conditions for the synthesis of BASP-ORCAs with narrow size distributions within the range of ˜25-40 nm, as well as high water solubility and relaxivity, m and N values from 5-10 and 15-30, respectively were screened (see Table 1). Gel permeation chromatography (GPC) revealed nearly quantitative MM-to-bottlebrush conversion as well as ≥85% bottlebrush-to-BASP conversion for all m and N values (FIGS. 7A-7B). The NP diameters as determined by dynamic light scattering (DLS) and transmission electron microscopy (TEM) ranged from ˜28 to ˜49 nm (see Table 1). A representative TEM image for the m=7.07 and N=20 BASP-ORCA (referred to as BASP-ORCA1) is provided in FIG. 2A. The hydrodynamic diameter (D_(h)) of this particle was 31±4 nm, which is suitable for extended in vivo circulation and tumor accumulation.

Characterization of BASP-ORCA Magnetic Properties

Electron paramagnetic resonance spectroscopy (EPR) was used to confirm the presence of chex in BASP-ORCAs, as well as to study the chex environment in BASP-ORCA1. The spin concentrations were ≥85% for all BASP-ORCAs. The height-normalized EPR spectra for BASP-ORCA1 and chex-MM are shown in FIG. 2B. The spectrum for BASP-ORCA1 is significantly broader than chex-MM, which is consistent with the larger and more rigid BASP nanostructure where the chex molecules are bound at the dense interface between the acetal crosslinker core and the PEG shell (FIGS. 1B and 2B). The BASP-ORCA1 spectrum was simulated using the procedure developed by Budil and Freed⁷⁴, which allows for characterization of the chex mobility in terms of the correlation time for rotational diffusion (τ). The spectrum was best fitted by superimposing two computed components (FIGS. 9A-9D): 22% corresponded to a relatively fast-moving nitroxide with r=0.2 ns while 78% corresponded to a very slow-moving nitroxide with r=10.0 ns. The faster-moving component likely corresponds to nitroxides that are furthest from the BASP-ORCA1 acetal core (FIG. 1B), while the slow-moving component corresponds to nitroxides that are close to and/or entangled within the rigid acetal core. Notably, the r of 10.0 ns measured for the slow component in BASP-ORCA1 is very large, which suggests that the majority of chex molecules are in a rigid environment. For comparison, in the previously reported chex-dendrimer ORCAs, TEMPO-labeled bottlebrush polymers, and BASPs, the largest r measured was ˜1 ns.

Next, the longitudinal (r₁) and transverse (r₂) relaxivities of these BASP-ORCAs were evaluated using a Bruker 7 T MRI scanner. The per-chex r₁ values as a function of m and N (Table 1) ranged from 0.27-0.53 mM⁻¹s⁻¹; they were not significantly increased compared to Rajca's chex-dendrimer and the chex-bottlebrush polymers. However, the per-chex r₂ values ranged from 2.90-7.40 mM⁻¹s⁻¹, which is ˜3.5-˜9.0-fold greater than the per-chex r₂ in the chex-bottlebrush polymers and ˜17-˜44-fold greater than 3-CP (Table 1). BASP-ORCA1 displayed a per-chex r₂ value of 4.67 mM⁻¹s⁻¹. Though this value was not the highest that was measured, BASP-ORCA1 was selected because it offered the best balance of high relaxivity, solubility (approximately 50 mg/mL, Table 1), and size for translation to biological studies. Given the number-average molar mass of BASP-ORCA1 as determined by gel permeation chromatography and static light scattering (M_(n)=4.75×10⁵ g/mol, Ð=1.32), it can be estimated that each BASP-ORCA1 particle contains an average of 92 chex groups. Based on this number, the estimated average molecular r₁ and r₂ values for BASP-ORCA1 are 37.6 mM⁻¹s⁻¹ and 428.8 mM⁻¹s⁻¹, respectively, which are greater than those for the commonly used FDA-approved Gd-based contrast agent Magnevist (r₁=3.1 mM⁻¹s⁻¹ and r₂=5.4 mM⁻¹s⁻¹ at 7 T) and iron-based NPs such as Feraheme (r₁=3.1 mM⁻¹s⁻¹ and r₂=68 mM⁻¹s⁻¹ at 7 T).^(75,76,77,78) The r₂/r₁ ratio for BASP-ORCA1 is approximately one-half that of Feraheme. Thus, BASP-ORCA1 should provide effective T₂-weighted MRI contrast enhancement.

MR phantom images of phosphate-buffered saline (PBS) solutions of BASP-ORCA1, chex-MM, and the previously reported chex-bottlebrush polymer at various chex concentrations (from 1 mM-4 mM chex) as well as a PEG-based BASP that lacks chex (at equivalent concentrations by mass as BASP-ORCA1) are provided in (FIG. 2C), along with images for “blank” PBS buffer. The T₁-weighted images for BASP-ORCA1, chex-MM, and chex-bottlebrush polymer are not obviously different while the T₂-weighted images clearly show a large decrease in signal for BASP-ORCA1 as concentration increases. The PEG-BASP with no chex shows no change in contrast as a function of concentration, which confirms that chex is required to observe any changes in image contrast.

The data presented above demonstrate that the high nitroxide density of BASP-ORCA1, which is a consequence of its unique crosslinked multi-layer nanostructure, affords an increased magnetization capability for r₂ enhancement. This finding is consistent with reports where nitroxides are utilized as magnetic catalysts for outer-sphere relaxation processes.^(79,80,81) Most importantly, the exceptionally high r₂ of BASP-ORCA1 overcomes one of the maj or limitations of nitroxide-based contrast agents: inherently low contrast.

Ascorbate Quenching Kinetics of BASP-ORCAs

As discussed above, nitroxide-based ORCAs typically suffer from rapid reduction to diamagnetic hydroxylamines under biologically relevant conditions. Amongst the many potential biological reducing agents, ascorbate (Asc) is known to play a major role in in vivo nitroxide reduction,^(82,83) and Asc-induced reduction can be amplified by glutathione (GSH). It was hypothesized that the rigid chex environment in the BASP-ORCAs could help to lower the rate of chex reduction. To test this hypothesis, EPR spectra for BASP-ORCA1 at various times were collected following exposure to 20 equivalents of Asc and 20 equivalents of GSH per nitroxide (both reagents were present in 10 mM concentrations). EPR spectra collected 1 minute, 40 minutes, and 180 minutes after exposure to these conditions are provided in FIG. 3A; the reduction in peak height as a function of time is indicative of nitroxide reduction. The normalized peak height of the EPR spectra are plotted versus time in FIG. 3B. Reduction kinetics data for the previous chex-bottlebrush polymers and chex-MM are provided for comparison. In contrast to the chex-bottlebrush and chex-MM samples, which both display an initial rapid chex reduction phase in the first hour, the reduction of chex in BASP-ORCA1 was significantly retarded with nearly 85% remaining after 1 hour, and 70% remaining after 3 hours (compared to 65% and 57%, respectively, for the chex-bottlebrush). Based on the integrated peak heights as a function of time, the second-order rate constants for BASP-ORCA1 reduction in the initial (first 10 minutes) and late (>1 hour) stages of the reduction process were calculated: k_(early)=0.0376 M⁻¹s⁻¹ and k_(late)≈0.00672 M⁻¹s⁻¹) (Table 2). Simulation revealed that the EPR spectra collected during the reduction process still consisted of a “fast” and a “slow” component (FIGS. 9A-9D). Interestingly, τ for the “fast” component remained constant at 0.2 ns, while τ for the “slow” component became increasingly larger with time (11.0 ns at 40 minutes and 13.2 ns at 180 minutes). Therefore, even after 3 hours there persists an extremely reduction resistant and slow moving nitroxide population, which suggests that BASP-ORCAs could be used for tumor MRI over longer timescales than have been possible with previous nitroxide contrast agents (vide infra).

Nitroxide Reduction Kinetics

TABLE 2 Kinetics of the reduction of nitroxides with 20-fold molar excess of ascorbate (Asc) and 0-25-fold molar excess of glutathione (GSH). Numerical fits to pseudo-first order rate equation (k′) peak height (PH) or integrated peak height (IPH) of the low-field EPR line. Late Initial Ki- Ki- Avg netics Ni- netics k × k × (>1 h) k × trox Asc. GSH (<1 h) k′ × 10⁴ 10⁴ Range k′ × 10⁴ Run Run Data Conc. Conc. Conc. Range 10⁴ (M⁻¹ (M⁻¹ of 10⁴ (M⁻¹ Compd No. Label used (mM) (mM) (mM) of fits (s⁻¹) R² s⁻¹) s⁻¹) fit (h) s⁻¹) R² s⁻¹) BASP- 1 JP1191 IPH   0.5 10 10 <1000 3.294 0.8795 329.4 366 ± 25  1.2-2.8 0.672 0.9923 67.2 ORCA1^(a) IPH*^(a) 3.40 0.9948 339.7 334 ± 55  0.586 0.9994 58.6 PH 0.836 0.8721 83.6 0.297 0.9943 29.7 2 JP1190 IPH   0.5 10 10 115-595 3.712 0.7664 371.2 IPH*^(a) 3.408 0.9910 340.8 PH 3.377 0.2923 33.77 JP1189 IPH   0.5 10 10 113-613 3.828 0.7646 382.8 IPH*^(a) 3.238 0.9863 323.7 PH 5.07 0.3068 50.7 4 JP1188 IPH   0.5 10 10 126-603 3.818 0.5387 381.8 IPH*^(a) 3.311 0.9938 331.1 PH 5.072 0.3366 50.72 1 YW982 IPH   0.5 10 5.0 177-897 3.27 0.9633 327.0 306^(b) chex- PH 3.42 0.9702 342.0 308^(b) bottle- brush 2 YW983 IPH   0.5 10 5.0  396-1019 2.85 0.9520 285.0 1.1-2.8 0.416 0.9216 41.6 PH 2.73 0.9895 273.0 0.386 0.9938 38.6 1 YW981 IPH   0.5 10 0.0 251-851 3.05 0.9439 305.0 296^(b) chex- PH 2.41 0.9808 241.0 254^(b) bottle- brush 2 YW985 IPH   0.5 10 0.0 278-878 2.86 0.9145 286.0 1.3-2.8 0.243 0.8838 24.3 PH 2.68 0.9775 268.0 0.196 0.9735 19.6 chex- 1 JP609  IPH   0.5 10 0.0  90-390 6.20 0.6609 620.0 603 ± 123 0.8-2.8 0.301 0.6847 30.1 den- drimer PH 6.17 0.9718 617.0  579 ± 59.6 0.354 0.9663 35.4 2 JP610  IPH   0.5 10 0.0 115-415 7.18 0.6743 718.0 PH 6.09 0.9336 609.0 3 JP611  IPH   0.5 10 0.0 126-426 4.72 0.7984 472.0 PH 5.10 0.9915 510.0 3-CP 1 JP899  IPH   0.2 4.0 5.0 <600 2.435 0.9997 608.8 608.0 ± 4.2  PH 2.361 0.9990 590.3 602.6 ± 25   2 JP8100 IPH   0.2 4.0 5.0 <600 2.438 0.9997 609.6 PH 2.410 0.9996 602.4 3 JP1101 IPH   0.2 4.0 5.0 <600 2.423 0.9998 605.6 PH 2.461 0.9996 615.2 3-CP 1 JP460  IPH   0.2 4.0 0.0 <3600 2.547 0.9996 636.8 625 ± 22  PH 2.504 0.9949 636.0 611 ± 44  2 JP461  IPH   0.2 4.0 0.0 <3600 2.498 0.9975 624.5 PH 2.396 0.9949 599.0 3 JP462  IPH   0.2 4.0 0.0 <3600 2.459 0.9999 614.8 PH 2.389 0.9961 597.3 >1 1.18  0.9952 295 ^(a)For BASP-ORCA1, double integration of entire EPR spectra gave initial rate constant k = 449 ± 23 M⁻¹s⁻¹, which is somewhat larger than the integrated peak height (IPH) value, k = 366 ± 25 M⁻¹s⁻¹; IPH* is the integrated peak height for the center line of the EPR spectrum. ^(b)For ORCA-Fluor, initial second order rate constants from 4 kinetic runs using 0-10 equivalents of GSH, k = 301 ± 20 and 281 ± 43 M⁻¹s⁻¹ for baseline corrected IPH and PH data. Data for chex-bottlebrush,⁹⁰ data for chex-dendrimer (baseline corrected) and late kinetics for 3-CP with Asc only, data for 3-CP with 20 equivalents of Asc and 25 equivalents of GSH,⁹² and data for 3-CP with Asc only⁹³ were reported elsewhere.

Fluorescence Properties of BASP-ORCAs

As noted above, Cy5.5 was also incorporated into these BASP-ORCAs (see FIG. 10 for BASP-ORCA1 absorption and emission spectra confirming the presence of Cy5.5) in order to simultaneously use NIRF as an imaging modality for comparison to MRI. Nitroxides are well-known to quench fluorescence via catalysis of non-emissive photophysical processes such as intersystem crossing. This quenching requires close interaction between the nitroxide and the fluorophore; the systems with the greatest quenching typically feature the nitroxide directly linked to the fluorophore via π bonds (i.e., electronic conjugation).^(84,85,86) Given the fact that chex and Cy5.5 are incorporated into BASP-ORCAs via two different macromonomers, and noting the limited mobility of chex in these nanoparticles, it was reasoned that Cy5.5 quenching would be minimal; therefore, Cy5.5 emission could be used as a fairly constant descriptor of particle concentration regardless of the extent of chex reduction.

To test this hypothesis, BASP-ORCA1 was exposed to a large excess of Asc (40 to 120 equivalents to chex) in water, and monitored the resulting Cy5.5 emission. In agreement with the expectation, only a 25±2% to 30±2% increase in fluorescence emission was observed (FIG. 3C). Moreover, addition of GSH (60 equivalents) as a co-reductant along with 60 equiv. of Asc gave only a 35±7% increase in fluorescence. Taken together, these data suggest that Cy5.5 fluorescence is only minimally quenched by chex in BASP-ORCA1. For comparison, exposure of the previously reported chex-bottlebrush polymer containing Cy5.5 to excess Asc or Asc+GSH led to 119±5% and 250±5% increases in fluorescence, respectively. Notably, the time required to achieve a fluorescence plateau varied significantly between BASP-ORCA1 (approximately 40 minutes) and the chex-bottlebrush polymer (a few minutes). Collectively, these data suggest that the BASP nanostructure provides greater steric shielding and isolation of chex and Cy5.5 compared to the analogous bottlebrush polymer.

In Vitro Cytotoxicity and In Vivo Gross Toxicity, Pharmacokinetics (PK), and Biodistribution (BD) of BASP-ORCA1 in Non-Tumor Bearing Mice

Motivated by BASP-ORCA1's unprecedented combination of properties, which include nanoscopic size (D_(h)=31±4 nm) and narrow size distribution, good water solubility, slow reduction kinetics, and exceptionally high r₂ relaxivity for an organic contrast agent, next the performance of this nanomaterial in biological assays was investigated. As discussed above, one potential advantage of ORCAs is their low toxicity. To investigate the toxicity of BASP-ORCA1, first in vitro human umbilical vein endothelial cell (HUVEC) and HeLa cell viability assays were conducted. In these assays, the cells were incubated with varied concentrations of BASP-ORCA1 for 72 h. Cell viability was determined by the CellTiter Glo assay (FIG. 11). The half-maximal inhibitory concentration (IC₅₀) of BASP-ORCA1, i.e., the concentration that led to 50% cell death, was 1.5 mg/mL (280 μM chex) and 4.5 mg/mL (830 μM chex) in HUVEC and HeLa cells, respectively. These results confirm that BASP-ORCA1 induces negligible in vitro cytotoxicity at practical concentrations. Next, the in vivo gross toxicity of BASP-ORCA1 was assessed. Healthy BALB/c mice were administered increasing doses (from 5 to 30 mg or 0.2 to 1.5 g/kg, respectively) of BASP-ORCA1 via tail vein injection. The animal body masses and behaviors were monitored over the course of 30 days. Loss of ≥10% body mass is generally considered to be a sign of unacceptable toxicity.^(87,88) As shown in FIG. 12, even the highest dose of BASP-ORCA1 (administered to n=4 animals) induced no significant decrease in body mass, which suggests that these particles are well-tolerated up to their solubility-limiting dose.

The pharmacokinetics (PK) and biodistribution (BD) of BASP-ORCA1 were monitored in healthy, non-tumor bearing BALB/c mice (n=3) using NIRF imaging (IVIS, Cy5.5 λ_(ex)/λ_(em)=640/700 nm). For PK analysis, blood samples were collected via cardiac puncture at various time points from 1 hour to 48 hours. Percent injected dose was plotted as a function of time (FIG. 13A). As is common for spherical nanoparticles, BASP-ORCA1 exhibited a two-phase clearance behavior, with an early distribution phase of ˜6 hours, followed by a steady elimination phase. Fitting the data presented in FIG. 13A with a standard two-compartment model yielded a blood compartment half-life for BASP-ORCA1 of 10 hours.⁸⁹ This long half-life is attributed to the nanoscale size of BASP-ORCA1, which limits renal clearance, and its PEGylated corona, which minimizes protein absorption and macrophage uptake. Consistent with these results and a plethora of studies on PEGylated nanoparticles, BD analysis revealed that a majority of BASP-ORCA1 accumulated in the liver, with increasing accumulation over 72 hours (FIG. 13B). Less, but significant, accumulation in the kidney and negligible accumulation in other tissues was observed (Note: fluorescence in extracted lung tissue is attributed to a high concentration of BASP-ORCA1 in the blood). Notably, fluorescence images of fecal samples (FIG. 13C) suggest that BASP-ORCA1 is ultimately cleared from the body via excretion.

BASP-ORCA1 BD in Tumor-Bearing Mice

Given the long circulation of BASP-ORCA1, it was hypothesized that this particle would passively accumulate in subcutaneous tumors following systemic injection. To test this hypothesis, a tumor model was established via subcutaneous injection of a mixture of 2.0×10⁶ lung carcinoma cells (A549, ATCC), Matrigel, and PBS buffer into a hind flank of NCR-NU mice (n=4). When the average tumor volume was ˜1 cm, BASP-ORCA1 was administered at a dose of 0.23 mmol chex/kg via tail vein injection. The mice were imaged 20 hours after administration. This choice of imaging time strikes a balance between allowing for sufficient tumor accumulation while limiting the extent of chex reduction in vivo. NIRF images indicated substantial tumor accumulation of BASP-ORCA1, which is consistent with other reports for PEGylated nanoparticles of similar size including the related drug-conjugated BASPs (FIG. 4A). Ex vivo BD data were consistent with the studies on non-tumor bearing BALB/c mice (i.e., liver accumulation and persistence in blood, FIGS. 13A and 13B) with the addition of significant tumor accumulation (FIG. 4C and FIG. 14).

In Vivo MRI and NIRF Imaging with BASP-ORCA1

The low toxicity, long circulation half-life, and tumor accumulation of BASP-ORCA1, along with its exceptional chex stability and relaxivity, suggested that this particle could be suitable for MRI of tumors following systemic injection and accumulation; a feat that has not yet been reported with ORCAs. Two groups of A459 tumor-bearing NCR-NU mice were administered different doses of BASP-ORCA1 via tail-vein injection: the “low dose” group (n=3) received 0.16s mmol chex/kg (0.8 g BASP-ORCA1/kg) while the “high dose” group (n=4) received 0.23 mmol chex/kg (1.2 g BASP-ORCA1/kg). The mice were anaesthetized and MR images were collected at various time points: 12 hours, 16 hours, and 20 hours post-injection for the low dose group and 20 hours post-injection for the high dose group. The images from each time point were compared to images collected before BASP-ORCA1 injection. FIG. 5A shows T₂-weighted images for a selected mouse from the low dose group imaged before BASP-ORCA1 injection (top row of images) and 20 hours (bottom row of images) after BASP-ORCA1 injection; from left-to-right the images correspond to progressive slices of the same animal in the z-axis with the tumor observed on the bottom right of each image. FIG. 5B shows an analogous set of images for a selected mouse from the high dose group. Contrast differences between the pre-injection and post-injection images can be observed at both dose levels, with greater contrast observed in the high dose animal. Whole animal images similarly revealed a clear difference in tumor contrast (FIG. 5C, arrows).

The percent negative contrast enhancement (i.e., signal reduction) before and after BASP-ORCA1 administration was quantified by image analysis (FIG. 5D). Signal reductions ranging from 14±2% to 16±2% (P≤0.05) were observed for the 12 hour to 20 hour time points in the low dose group (FIG. 5D, low dose bars). In the high dose group, a 24±2% (P≤0.001) signal reduction was observed 20 hours after BASP-ORCA1 administration (FIG. 5D, high dose right bar). The clear BASP-ORCA1 dose-response effect suggests that the observed contrast differences between pre- and post-injection are due to accumulation of BASP-ORCA1 in the tumors. Keeping in mind that MRI phantoms revealed no observable contrast enhancement for PEG-BASPs that lack chex (FIG. 2C), these MRI data imply that 20 hours following injection there is a sufficient concentration of non-reduced chex (i.e., chex radicals) present on the BASP-ORCA1 to impart contrast. To confirm the presence of chex radicals in the tumors, the same mice that were imaged by MRI were sacrificed 21 hours after BASP-ORCA1 administration and their tissue homogenates and blood were analyzed by EPR spectroscopy (FIG. 6A). From these spectra, the radical concentration per g protein in each tissue sample, the latter obtained via a bicinchoninic acid assay (BCA), of the tissue homogenate, was evaluated and normalized by the radical concentration per g protein in muscle tissue (FIG. 6B). In agreement with the MRI data, the concentration of free radicals in the tumor was quite high 22 hours after BASP-ORCA1 injection; the measured value of 0.25 μmol±0.04 chex/g of protein corresponds to 4.5% of the injected dose of chex radicals (Note: this value does not include any chex radicals that were reduced, and thus the percent injected dose of BASP-ORCA1 in the tumor is likely larger than 4.5%). Moreover, consistent with the in vivo NIRF imaging results (vide supra), relatively high radical concentrations were observed in the liver and kidney, which suggests that the clearance of BASP-ORCA1 proceeded mostly through these organs. Notably, the murine liver contains a high concentration of Asc (millimolar range); the observation of radicals in the liver 22 hours after injection is further evidence of the extremely stable nature of the chex units in BASP-ORCA1 (Note: in the previous chex-bottlebrush polymers, there was very little chex radical in the liver following 30 minutes and none after 24 hours). A high chex concentration was also observed in the heart, which is in accord with a long blood compartment half-life and is consistent with the PK data obtained by NIRF imaging. Finally, NIRF imaging of these homogenates provided fluorescence radiant efficiencies that were in good agreement with the spin concentrations (FIG. 6B), which suggests that the chex radicals and Cy5.5 dyes are still co-localized within the BASP-ORCA1 construct 22 hours after injection. Unlike the previous chex-bottlebrush polymers, which displayed dramatic increases in fluorescence as chex was reduced, the signal uniformity offered by BASP-ORCA1 provides for straightforward multi-modal confirmation of BD.

BASP-ORCA1 is the first nitroxide MRI contrast agent capable of providing significant contrast 20 hours after injection, which is a testament to its unique structural features that combine optimal size for tumor accumulation with a high nitroxide density and stability. To set these results in context, the data outlined herein was compared to recent literature examples of MRI-contrast agents the rely on metals to achieve tumor imaging following systemic administration. For example, Kataoka and coworkers recently reported on a new class of Gd-based nanoparticles (T₁ contrast agents) for MRI of tumors. In their study, a ˜40% contrast enhancement (at 0.05 mmol Gd/kg iv dose) was observed plateauing 4 hours following injection into mice bearing subcutaneous C26 tumors. Notably, commercially available Gd-DTPA, which is a small molecule, exhibited negligible contrast enhancement (at 0.23 mmol Gd/kg iv dose) after 4 hours. This example highlights the importance of a nanoparticle system for extended circulation and tumor imaging, though the impact of Gd nanoparticle accumulation in tissues would need to be addressed prior to clinical translation. In another example, the same group reported novel Fe-based nanoparticles (T₂ contrast agents) for tumor imaging in a similar murine model (subcutaneous C26 tumors). Here, an approximately 25% contrast difference was observed 24 hours following intravenous administration of 0.45 mg Fe/kg. Notably, less than 10% contrast enhancement was observed using commercially available Resovist® (at 0.45 mg Fe/kg intravenous dose).^(Error! Bookmark not defined). It should be noted that the instrument parameters used to obtain T₂-weighted images in this work were similar to those used in the studies described herein; thus, these results for BASP-ORCA1 are on par with recently reported nanoparticle MRI contrast agents that rely on metals to achieve contrast.

Conclusion

In conclusion, a nitroxide-nanoparticle MRI contrast agent—BASP-ORCA1—that enables simultaneous MRI and NIRF imaging in vivo over timescales suitable for tumor imaging following systemic injection was developed herein. BASP-ORCA1 addresses the two major challenges that have historically limited nitroxide-based organic radical contrast agents for MRI: low relaxivity and poor stability. These functions were made possible by the brush-arm star polymer (BASP) nanostructure, which enables the placement of chex nitroxides at the interface between a rigid poly(acetal) core and a hydrophilic PEG shell. Altogether, BASP-ORCA1 displayed unprecedented per-nitroxide and per-molecule transverse relaxivities for organic radical contrast agents, exceptional stability, high water solubility, low in vitro and in vivo toxicity, and a long blood compartment half-life. These features combined to facilitate the imaging of subcutaneous tumors in mice 20 hours after tail-vein injection, providing contrast enhancements on par with commercial and literature examples of metal-based contrast agents. This work suggests that organic radicals can be viable alternatives to metal-based MRI contrast agents, and sets the stage for the development of theranostic systems that combine organic radical contrast agents with therapeutic payloads to achieve simultaneous tumor imaging and drug delivery without concerns over long-term accumulation of metals.

Materials, General Methods, and Instrumentation

All reagents were purchased from commercial suppliers and used without further purification unless stated otherwise. Grubbs 3^(rd) generation bispyridyl catalyst,⁷³ macromonomers (MMs) chex-MM,² Cy-MM,⁹⁰ PEG-MM and cross-linker Acetal-XL were prepared according to literature procedures. Size exclusion chromatography (SEC) analyses were performed on an Agilent 1260 Infinity setup with two Shodex KD-806M columns in tandem and a 0.025 M LiBr DMF mobile phase run at 60° C. The differential refractive index (dRI) of each compound was monitored using a Wyatt Optilab T-rEX detector, and the light scattering (LS) signal was acquired with a Wyatt Dawn Heleos-II detector. Column chromatography was carried out on silica gel 60F (EMD Millipore, 0.040-0.063 mm).

Dynamic light scattering (DLS) measurements were performed using a Wyatt Technology Mobius DLS instrument. Samples were prepared at 1.0 mg/mL in either nanopure water (MilliQ), PBS buffer, or 5% glucose solution (in nanopure water). The resulting solutions were passed through a 0.4 μm Nalgene filter (PES membrane) into disposable polystyrene cuvettes, which were pre-cleaned with compressed air. Measurements were made in sets of 10 acquisitions, and the average hydrodynamic diameters were calculated using the DLS correlation function via a regularization fitting method (Dynamics 7.4.0.72 software package from Wyatt Technology).

TEM images were acquired using a FEI Tecnai Multipurpose TEM (G2 Spirit TWIN, 12 kV) at the MIT Center for Materials Science and Engineering. Samples were prepared as follows: 5 μL of a 1.0 mg/mL aqueous solution of BASP-ORCA was pipetted onto a carbon film-coated 200-mesh copper grid (Electron Microscopy Sciences) placed on a piece of parafilm. Next, the solution was carefully absorbed at the base of the droplet using the edge of a Kimwipe, leaving behind the nanoparticles on the TEM grid. The samples were then negatively stained by adding a drop of 2 wt % uranyl acetate (Electronic Microscopy Sciences). After 3 min, the residual uranyl acetate solution was carefully absorbed onto a Kimwipe, and the samples were allowed to dry completely.

Excitation/emission spectra and fluorescence measurements were acquired using a Tecan Infinite® 200 Pro plate reader. Electron Paramagnetic Resonance (EPR) spectra were acquired at the University of Nebraska using a Bruker CW X-band spectrometer equipped with a frequency counter. The spectra were obtained using a dual mode cavity; all spectra were recorded using an oscillating magnetic field perpendicular (TE₁₀₂) to the swept magnetic field. DPPH powder (g=2.0037) was used as a g-value reference.

Relaxivity Measurements by MRI

Phantom MRI data were acquired in a 12 cm outer diameter birdcage transceiver for imaging in a 20 cm bore Bruker 7 T Avance III MRI scanner. Samples at varying concentrations (0 up to 5 mM) in PBS buffer were loaded into the wells of a 384-well clear polystyrene plate (Thermo Scientific Nunc), which had been pre-cut in half to optimally fit the coil. Unused wells were filled with PBS buffer. 2 mm slices were imaged through the samples with the field of view of 5×5 cm and the data matrices were 256×256 points. Longitudinal (r₁) and transverse (r₂) relaxivity measurements were acquired using multi-spin multi-echo (MSME) sequences (flip angle=180°). r₁; TE=12 ms, TR=300, 350, 400, 450, 500, 600, 800, 1000, 1200, 1500, 3000, 5000, 10000 ms. r₂; TR=5000 ms, TE=12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144, 156, 168, 280, 192, 204, 216, 228, 240, 252, 264, 276, 288, 300, 312, 324, 336, 348, 360 ms. Custom routines written in Matlab (Mathworks, Natick, Mass.) were used to reconstruct the images and compute relaxation time constants by fitting image intensity data to exponential decay curves.

Kinetics of Nitroxide Quenching by EPR Spectroscopy

A solution was prepared with ascorbic acid (Asc), sodium phosphates (<30 ppm transition metals), sodium hydroxide and diethylenetriaminepentaacetic acid (DTPA, ˜0.1% (mol/mol) to sodium phosphates) at pH 7.4. Reduced L-GSH was then dissolved to provide the Asc/GSH solution. BASP-ORCA solution was prepared in phosphate buffer, which was made from sodium phosphates and DTPA (˜0.1% (mol/mol) to sodium phosphates) at pH 7.4. Equal volumes of the freshly prepared 1 mM (in nitroxide) sample solution and 20 mM Asc/GSH solution were combined and vortexed for 6 seconds, and then added to a 2 mm OD EPR tube. Kinetic studies were performed on 0.5 mM nitroxide solution in the presence of 125 mM sodium phosphates, 10 mM Asc, and 10 mM GSH. The peak height of the low-field line of the triplet was measured as a function of time. Microwave power was kept under 6.5 mW and the temperature was controlled at 295 K with a nitrogen flow system.

Computational Analysis of Nitroxide Quenching by EPR Spectroscopy

The EPR spectra are constituted by a “fast” and a “slow” component. From visual inspection, it was clear that the slow component was changing from one to another sample, while the fast one showed an almost equivalent line shape in the three spectra. Therefore, first a computation (program by Budil&Freed⁷⁴) of the fast component to be subtracted from the three spectra to obtain a reliable line shape for the slow components was employed. This succeeded for the fast component shown in FIG. 9A (the subtracted experimental line in black and the computed line is in red). The main parameters used for the computation are shown in the figure and described below. Subtraction of this fast component from the overall spectra produced the three slow components shown in FIGS. 9B, 9C and 9D for 1 min, 40 min, and 180 min, respectively (in FIGS. 9A-9D the spectra are normalized in height). Their computations are shown as well, together with the main parameters used for computation and analysis. The following parameters were calculated.

The g_(ii) components for the coupling between the electron spin and the magnetic field (accuracy from computation±0.0002). The starting values, which were used in previous studies⁹¹ using nitroxide radicals, are 2.009, 2.006, 2.003, for g_(xx), g_(yy), and g_(zz), respectively. It was found that these values worked for the computations of the fast component and for the t=1 minute slow component; however, for computing the slow components of t=40 minutes and 180 minutes it was necessary to decrease the g_(zz) values to 2.0025 and 2.002, respectively. This observation indicated an increased structural anisotropy of the nitroxide labels from 1 minute to 40 minutes to 180 minutes.

The A_(ii) components for the coupling between the electron spin and the nitroxide-nitrogen nuclear spin (accuracy from computation±0.5 G). These parameters increase with an increase in the environmental polarity of the nitroxide. Mainly, as done in previous studies,⁹¹ the A, and A_(yy) values were maintained constant (6 G) and only A_(zz) was changed. The polarity was found to be slightly lower for the fast component (A_(zz)=35 G) than for the slow one (A_(zz)=36 G); it was constant for the different slow components.

The correlation time for rotational diffusion of the radical, z (accuracy from computation±0.05 ns). This parameter increases with an increase in the local viscosity around the nitroxide group and with a decrease in the rotational mobility of the nitroxide. The local viscosity largely increased (the mobility decreased) from the fast component to the slow ones and it also increased (the mobility decreased) from 1 minute (10 ns) to 40 minutes (11 ns) to 180 minutes (13.2 ns). Notably, by performing a subtraction procedure using the double integrals of the components of the spectra, it was found that the fast component was contained in all the three spectra in almost the same relative percentage, that is, about 20% (the accuracy in this percentage is about 1%).

The line width (accuracy from computation±0.1 G), which measures spin-spin interactions due to a high local concentration of paramagnetic species (like colliding nitroxide groups in fast motion, or nitroxides bound in close proximity in slow motion). The line width was quite high for all samples, indicating a high local concentration of nitroxides, but it was the highest (7.6 G) for the slow component of the t=1 minute sample, and it decreased at 40 minutes (5.5 G) and further decreased at 180 minutes (4.2 G). The latter value is even smaller than the line width of the fast component (4.8 G).

Fluorimetry

Fluorescence analysis was performed using a Tecan Infinite® 200 Pro plate reader. Absorption/emission spectra of BASP-ORCA1 were acquired to determine λ_(ex/em), which were 640 nm and 705 nm respectively (as expected for the dye used in these studies: Cyanine5.5). Absorption spectra were acquired using a 1 nm wavelength step size at 9 nm bandwidth; emission spectra were obtained using λ_(ex) of 640 nm, a 5 nm wavelength step size, and 10 nm bandwidth. To examine the effect of nitroxide-quenching on fluorescence emission intensity, samples were prepared in 96-well plates (Corning, n=3) by mixing 50 μL of 5 mg BASP-ORCA1/mL solution with 50 μL of Asc/GSH solution with one of the following compositions: 120 equivalents (eq, with respect to chex) Asc, 60 eq Asc, 40 eq Asc, and 60 eq Asc+60 eq GSH. Control samples (n=3) were prepared by mixing 50 μL of 5 mg BASP-ORCA1/mL solution with 50 μL of PBS. Fluorescence intensity was monitored continuously for 2 hours; a plateau was typically reached within 40-50 min.

Cell Culture

A549 and HeLa cells (ATCC) were cultured in DMEM media (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, VWR) and 1% penicillin/streptomycin (Thermo Fisher Scientific). Human umbilical vein endothelial cells (HUVEC, Lonza) were cultured in EGM⁺ media (Lonza) supplemented with 1% penicillin/streptomycin. All cells were housed in 5% CO₂ humidified atmosphere at 37° C.

In Vitro Cell Viability

HUVEC cells were plated at 5,000 cells per well (in 100 μL) in 96-well collagen-coated plates (Corning) and allowed to adhere overnight. The media was then replaced with fresh media containing BASP-ORCA1 at various concentrations. The plate was incubated for 72 hours, and cell viability was then determined using the CellTiter-Glo assay (Promega). HeLa cells were plated in 96-well plates (Corning) and cytotoxicity was studied following the same experimental procedure used for HUVEC cells.

Animal Usage

All experiments involving animals were reviewed and approved by the MIT Committee for Animal Care (CAC). BALB/c mice (female, 8-12 weeks old, Taconic) were used for in vivo toxicity, pharmacokinetic studies, and biodistribution (n=3). NCR-NU nude mice (female, 8-12 weeks old, Taconic) were used for in vivo MRI, NIRF imaging, and biodistribution (n=3). All animals received an alfalfa-free diet (TestDiet) at least 2 weeks prior to the start of the studies to minimize auto-fluorescence.

In Vivo Toxicity

Solutions containing 5.0-30 mg of BASP-ORCA1 in 5% glucose were prepared, passed through sterile 0.2 μm filter (Nalgene, PES membrane), and administered into BALB/c mice via tail vein injection. The mice were monitored over a period of 30 days. Initial injections were performed in one mouse for each dose, all of which appeared to be well-tolerated. The highest dose (30 mg) was then administered to another set of mice (n=3). No adverse physical effects and/or significant weight losses were observed.

In Vivo MR and NIRF Imaging Instrumentation

All imaging experiments were performed at the Koch Institute for Integrative Cancer Research at MIT. In vivo MRI was acquired using a Varian 7T/310/ASR-whole mouse MRI system. Scans were collected with respiratory gating (PC-SAM version 6.26 by SA Instruments Inc.) to avoid confounding noise due to chest movement. The respiratory rate and animal temperature were closely monitored during image collection. Coronal T2WIs were collected using the fast spin echo multiple slices pulse sequence with T_(R)=4000 ms; T_(E(eff))=48 ms; ETL=8; FOV=100×50 mm²; 512×256 matrix and 2 averages over 12 slices of 1 mm thickness and 0 mm gap. Axial T2WIs were collected using the fast spin echo multiple slices pulse sequence with T_(R)=4⁰⁰⁰ ms; T_(E(eff))=48 ms; ETL=8; FOV=45×45 mm²; 256×256 matrix and 2 averages over 10-16 (to capture entire tumor) slices of 1 mm thickness and 0 mm gap.

In vivo NIRF imaging was performed on an IVIS Spectrum-bioluminescent and fluorescent imaging system (Xenogen). Epi-fluorescence imaging was acquired through excitation of the Cy5.5 fluorophore (λ_(ex)/λ_(em)=640/700 nm, exposure time 2-10 seconds) present in BASP-ORCA1.

Pharmacokinetics (PK) and Biodistribution (BD) Studies

BASP-ORCA1 doses (5.0 mg in 5% glucose) were prepared, passed through sterile 0.2 μm filters, and injected into BALB/c mice (groups of n 3). Blood samples were taken at 1, 3, 6, 24, and 48 hours via cardiac puncture after euthanization in a CO₂ chamber. The blood samples were subjected to fluorescence imaging (IVIS, Cy5.5 λ_(ex/em)=640/700 nm, Xenogen) for analysis of blood-compartment PK. For BD, organs from these BALB/c mice were harvested and subjected to fluorescence imaging (IVIS, Cy5.5 λ_(ex/em)=640/700 nm, Xenogen).

In Vivo MR and NIRF Imaging in Tumor-Bearing Mice

A549 cells were cultured in DMEM media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in 5% CO₂ humidified atmosphere (37° C.) to a final concentration of 20%. Cells were then harvested, mixed with Matrigel and sterile pH 7.4 PBS buffer (1:1), filtered through sterile 0.2 μm filters, and injected subcutaneously (2.0×10⁶ cells) into the hind flank of NCR-NU mice. Tumor growth was monitored for 2-4 weeks until appropriate cumulative diameters (˜1 cm) were achieved.

MRI and NIRF images were acquired for each animal (n=3-4) before injections. BASP-ORCA1 doses (0.16 mmol chex/kg or 0.23 mmol chex/kg in 5% glucose) were prepared, passed through a sterile 0.2 μm filter, and administered to the tumor-bearing mice via tail vein injection. Tumor imaging was done at pre-determined time points; at the last imaging time point, mice were immediately euthanized in a CO₂ chamber, and organs were collected, imaged by NIRF, and stored in dry ice for EPR analysis.

Ex Vivo EPR Spectroscopy

Harvested organs were shipped on dry ice to the University of Nebraska, where they were stored on dry ice. For EPR sample preparation, each tissue sample, one at a time, was rapidly thawed and transferred to a weighed vial; 900 μL of PBS buffer (0.5 mM, pH 7.2) was then added. The mixture was put into an ice-water bath and homogenized with a rotor stator homogenizer, then pipetted into a 4-mm outer diameter EPR sample tube. The samples were degassed by sonication as needed (for instance, when gas bubbles were visible). The EPR tube was capped, sealed with parafilm, and stored briefly in acetone/dry ice bath before spin concentration measurements.

Spin concentrations of nitroxide radicals in tissues (mol chex per g protein; Note: see below for details of protein content determination) were measured at −30° C. (243.2 K) to increase signal-to-noise of the aqueous samples. Measurements of tissue samples were alternated with that of the spin concentration reference (see next paragraph) and g-value reference (2,2-diphenyl-1-picrylhydrazyl powder was used as the g-value reference). For tissue samples with low signal-to-noise, the cavity background was recorded with identical parameters, including number of scans and receiver gain. Typical parameters were as follows: microwave attenuation-20 dB, modulation amplitude-5 Gauss, spectral width-300 Gauss, resolution-512 points, conversion-40.96, time constant-10.24, and sweep time-20.97 seconds. These parameters were kept identical for the tissues, references, and cavity backgrounds. The number of scans (8-256) and receiver gain were adjusted as needed for each sample.

The reference for spin concentration was 0.50 mM Proxyl in PBS (pH 7.2). This reference was always stored in dry ice, except during measurements, and occasionally re-checked for spin concentration decay.

Protein Content Determination

The protein content of tissue homogenate samples was determined using the BCA Protein Assay Kit (ThermoFisher Scientific). These protein contents were then used as a normalizing parameter to compare nitroxide spin concentration and NIRF signal (FIG. 6B).

Ex Vivo NIRF Imaging

To acquire BD, the collected organs and organ homogenates were subjected to NIRF imaging following the same aforementioned experimental procedure as for in vivo NIRF imaging. Furthermore, tissue homogenate samples were transferred into a 96-well plate and imaged for the correlation of NIRF signal and spin concentration.

In Vivo MRI Data Analysis

Signal intensities pre- and post-injection were compared only using slices where tumors and muscle were clearly visible. Using ImageJ software, a region of interest (ROI) around each component was manually drawn. The average signal intensity and area of the ROI were measured; these data were then normalized against the signal intensity of the muscle tissue. Signal intensity was acquired by multiplying area and normalized signal intensity. This process was repeated for all relevant slices for a given organ; the sum of these signal intensities was then calculated and divided for the total area, affording the volume-averaged signal intensity. Signal enhancement by BASP-ORCA1 was quantified by comparing the volume-averaged signal intensities pre- and post-injection.

Procedure for BASP-ORCA Synthesis

Representative Procedure for BASP-ORCA Synthesis with Brush Length of 7.07 (m) and 20 Equivalents (N) of Cross-Linker (BASP-ORCA1, m=7.07, N=20)

All BASP-ORCA syntheses were performed in a glovebox under N2 atmosphere; however, similar results are expected under ambient conditions. All ROMP reactions followed the same general procedure, which was modified from literature examples.

To a 4 mL vial, a suspension of Acetal-XL (15.6 mg, 26.8 μmol, 20.0 eq) in THF (268.0 μL, 0.1 M Acetal-XL) was prepared. To a second 4 mL vial containing a stir bar, chex-MM (35.0 mg, 9.4 μmol, 7.0 eq) was added; Cy-MM was then added from a premade 12.5 mg/mL solution in THF (30.6 μL, 0.094 μmol, 0.07 eq). To a third vial, a solution of Grubbs 3^(rd) generation bispyridyl catalyst (Grubbs III, 0.02 M in THF) was freshly prepared. THF (91.8 μL) was then added to the MM vial, followed by the addition of Grubbs III solution (67.0 μL, 1.3 mol, 1.0 eq) to give the desired MM:Grubbs III ratio of 7.07:1 (1 mol % of the Cy-MM), while achieving a total MM concentration of 0.05 M, affording a dark blue solution. The reaction mixture was allowed to stir for 30 minutes at room temperature before an aliquot (˜5 μL) was taken out and quenched with 1 drop of ethyl vinyl ether for GPC analysis. The Acetal-XL suspension was then added dropwise (in aliquots of 5 eq, or ˜70 μL, every 5 minutes) over the course of 20 minutes into the MM vial, and the polymerizing mixture was allowed to stir for 6 hours at room temperature, affording a dark blue solution. To quench the polymerization, a drop of ethyl vinyl ether was added. The reaction mixture was transferred to an 8 kD molecular weight cutoff dialysis tubing (Spectrum Laboratories) in 10 mL nanopure water, and the solution was dialyzed against water (500 mL×3, solvent exchange every 6 h). The solution of BASP-ORCA was then lyophilized to afford a blue solid.

Other BASP compositions were prepared as follows: MM:Grubbs III ratios of 9.99:1, 7.07:1, or 5.05:1 (m values). Acetal-XL were used in 15, 20, or 30 equivalences (N values). PEG-BASP, which contained no chex-MM, was prepared in an analogous manner to BASP-ORCAs using a PEG-MM lacking chex. Chex-bottlebrush was prepared as previously described.⁹⁰

EQUIVALENTS AND SCOPE

In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

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1. A brush-arm star polymer comprising at least 100 repeating units selected from Formula (I) and Formula (II):

or a salt thereof, wherein: each of A, A¹, and B is independently C₁-C₁₂ alkylene, C₂-C₁₂ alkenylene, C₂-C₁₂ alkynylene, or C₁-C₁₂ heteroalkylene, C₂-C₁₂ heteroalkenylene, C₂-C₁₂ heteroalkynylene, wherein each alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene is optionally substituted with 1-24 independently selected R¹; X is an imaging agent; P is alkylene, heteroalkylene, or polymer; L is a bond, —O—, —S—, —S—S—, alkylene, alkenylene, alkynylene, heteroalkylene, alkylene-arylene-alkylene, (C₀-C₁₂ heteroalkylene)-arylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ alkylene)-arylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ heteroalkylene)-arylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ alkylene)-heteroarylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heteroarylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heteroarylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ alkylene)-heterocyclylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heterocyclylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-aryl-(C₀-C₁₂ heteroalkylene), or (C₀-C₁₂ heteroalkylene)-heterocyclylene-(C₀-C₁₂ heteroalkylene), wherein each alkylene, alkenylene, alkynylene, heteroalkylene, arylene, heteroarylene, or heterocyclylene is optionally substituted with 1-24 independently selected R¹, and combinations thereof; each R¹ is independently alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR^(A), —N(R^(A))₂, —NR^(A)C(O)R^(A), —NR^(A)C(O)OR^(A), —NR^(A)C(O)N(R^(A))₂, —C(O)N(R^(A))₂, —C(O)R^(A), —C(O)OR^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂, —SR^(A), or —S(O)_(m)R^(A); each R^(A) is independently hydrogen, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, or C₁-C₆ haloalkyl; each of a and b are independently an integer between 1 and 10000, inclusive; each of “1”, “2”, “3”, “4”, “5”, and “6” is independently a terminal group selected from the group consisting of hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted acyl, optionally substituted hydroxyl, optionally substituted amino, and optionally substituted thio; or represents a bond to a structure of Formula (I) or Formula (II); y is an integer between 1 and 100, inclusive; and m is 1 or
 2. 2. The brush-arm star polymer of claim 1, wherein P is polymer selected from the group consisting of polyethers, polyesters, polyacrylamides, polycarbonates, polysiloxanes, polyfluorocarbons, polysulfones, polystyrene, and polyethylene glycol with a molecular weight ranging from about 200 g/mol to about 6000 g/mol.
 3. (canceled)
 4. The brush-arm star polymer of claim 1, wherein B is C₁-C₁₂ alkylene, optionally substituted with 1-24 independently selected R¹; R¹ is alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR^(A), —N(R^(A))₂, —NR^(A)C(O)R^(A), —NR^(A)C(O)OR^(A), —NR^(A)C(O)N(R^(A))₂, —C(O)N(R^(A))₂, —C(O)R^(A), —C(O)OR^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂, —SR^(A), or —S(O)_(m)R^(A); each R^(A) is independently hydrogen, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, or C₁-C₆ haloalkyl; and m is 1 or
 2. 5. The brush-arm star polymer of claim 1, wherein A is C₁-C₁₂ alkylene, optionally substituted with 1-24 independently selected R¹; R¹ is alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR^(A), —N(R^(A))₂, —NR^(A)C(O)R^(A), —NR^(A)C(O)OR^(A), —NR^(A)C(O)N(R^(A))₂, —C(O)N(R^(A))₂, —C(O)R^(A), —C(O)OR^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂, —SR^(A), or —S(O)_(m)R^(A); each R^(A) is independently hydrogen, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, or C₁-C₆ haloalkyl; and m is 1 or
 2. 6. (canceled)
 7. The brush-arm star polymer of claim 1, wherein L is independently selected from

wherein: q, p, and o are independently an integer between 0 and 20, inclusive.
 8. (canceled)
 9. The brush-arm star polymer of claim 1, wherein the imaging agent X is a chelated metal, inorganic compound, organic compound, or salt thereof. 10-11. (canceled)
 12. The brush-arm star polymer of claim 1, wherein the imaging agent X is of the formula:

13-14. (canceled)
 15. The brush-arm star polymer of claim 1, wherein the repeating unit of Formula (I) is of formula:

or a salt thereof, wherein: each of A, A¹, and B is independently C₁-C₁₂ alkylene, C₂-C₁₂ alkenylene, C₂-C₁₂ alkynylene, or C₁-C₁₂ heteroalkylene, C₂-C₁₂ heteroalkenylene, C₂-C₁₂ heteroalkynylene, wherein each alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene is optionally substituted with 1-24 independently selected R¹; X is an imaging agent; P is alkylene, heteroalkylene, or polymer; L is a bond, —O—, —S—, —S—S—, C₁-C₁₂ alkylene, C₂-C₁₂ alkenylene, C₂-C₁₂ alkynylene, C₁-C₁₂ heteroalkylene, (C₀-C₁₂ alkylene)-arylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-arylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ alkylene)-arylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ heteroalkylene)-arylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ alkylene)-heteroarylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heteroarylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heteroarylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ alkylene)-heterocyclylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heterocyclylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-aryl-(C₀-C₁₂ heteroalkylene), or (C₀-C₁₂ heteroalkylene)-heterocyclylene-(C₀-C₁₂ heteroalkylene), wherein each alkylene, alkenylene, alkynylene, heteroalkylene, arylene, heteroarylene, or heterocyclylene is optionally substituted with 1-24 independently selected R¹, and combinations thereof; each R¹ is independently alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR^(A), —N(R^(A))₂, —NR^(A)C(O)R^(A), —NR^(A)C(O)OR^(A), —NR^(A)C(O)N(R^(A))₂, —C(O)N(R^(A))₂, —C(O)R^(A), —C(O)OR^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂, —SR^(A), or —S(O)_(m)R^(A); each R^(A) is independently hydrogen, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, or C₁-C₆ haloalkyl; y is an integer between 1 and 100, inclusive; and m is 1 or
 2. 16. The brush-arm star polymer of claim 1, wherein the repeating unit is of formula:


17. (canceled)
 18. The brush-arm star polymer of claim 1, wherein the repeating unit of Formula (II) is of formula:

a salt thereof, wherein: L is a bond, —O—, —S—, —S—S—, C₁-C₁₂ alkylene, C₂-C₁₂ alkenylene, C₂-C₁₂ alkynylene, C₁-C₁₂ heteroalkylene, (C₀-C₁₂ alkylene)-arylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-arylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ alkylene)-arylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ heteroalkylene)-arylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ alkylene)-heteroarylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heteroarylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heteroarylene-(C₀-C₁₂ heteroalkylene), (C₀-C₁₂ alkylene)-heterocyclylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-heterocyclylene-(C₀-C₁₂ alkylene), (C₀-C₁₂ heteroalkylene)-aryl-(C₀-C₁₂ heteroalkylene), or (C₀-C₁₂ heteroalkylene)-heterocyclylene-(C₀-C₁₂ heteroalkylene), wherein each alkylene, alkenylene, alkynylene, heteroalkylene, arylene, heteroarylene, or heterocyclylene is optionally substituted with 1-24 independently selected R¹, and combinations thereof; each R¹ is independently alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, nitro, —OR^(A), —N(R^(A))₂, —NR^(A)C(O)R^(A), —NR^(A)C(O)OR^(A), —NR^(A)C(O)N(R^(A))₂, —C(O)N(R^(A))₂, —C(O)R^(A), —C(O)OR^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂, —SR^(A), or —S(O)_(m)R^(A); each R^(A) is independently hydrogen, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, or C₁-C₆ haloalkyl; and m is 1 or
 2. 19. The brush-arm star polymer of claim 1, wherein the repeating unit is of formula:


20. The brush-arm star polymer of claim 1, wherein the ratio of repeating unit of Formula (I) and repeating unit of Formula (II) is between about 1:20 to about 20:1, respectively.
 21. (canceled)
 22. A method of producing a brush-arm star polymer comprising (a) reacting one or more macromonomers including an imaging agent with a metathesis catalyst to form a living polymer; and (b) mixing a crosslinker with the living polymer. 23-46. (canceled)
 47. A composition comprising the polymer of claim 1 and a pharmaceutically acceptable excipient.
 48. (canceled)
 49. A kit comprising a polymer of claim 1, and instructions for use.
 50. A method of imaging a subject, the method comprising steps of: administering to a subject the polymer of claim 1; and acquiring an image.
 51. A method of performing magnetic resonance imaging (MRI) of a subject, the method comprising steps of: administering to a subject the polymer of claim 1; and acquiring a magnetic resonance image.
 52. A method of performing near-infrared fluorescence (NIRF) imaging of a subject, the method comprising steps of: administering to a subject the polymer of claim 1; and acquiring a near-infrared fluorescence image.
 53. A particle comprising a polymer of claim
 1. 54. A nanoparticle comprising a polymer of claim
 1. 55. (canceled) 